EP2947070A1 - Multi-targeted ubenimex prodrug derivative and preparation method and use thereof - Google Patents
Multi-targeted ubenimex prodrug derivative and preparation method and use thereof Download PDFInfo
- Publication number
- EP2947070A1 EP2947070A1 EP14807120.2A EP14807120A EP2947070A1 EP 2947070 A1 EP2947070 A1 EP 2947070A1 EP 14807120 A EP14807120 A EP 14807120A EP 2947070 A1 EP2947070 A1 EP 2947070A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- compound
- ubenimex
- give
- targeted
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229950009811 ubenimex Drugs 0.000 title claims abstract description 70
- 229940002612 prodrug Drugs 0.000 title claims abstract description 47
- 239000000651 prodrug Substances 0.000 title claims abstract description 47
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 title claims abstract 22
- 238000002360 preparation method Methods 0.000 title claims description 52
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 88
- 150000001875 compounds Chemical class 0.000 claims abstract description 69
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 40
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 35
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 80
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 69
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 45
- 229960002949 fluorouracil Drugs 0.000 claims description 44
- 239000003814 drug Substances 0.000 claims description 37
- 229940079593 drug Drugs 0.000 claims description 37
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 31
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 30
- 125000001424 substituent group Chemical group 0.000 claims description 29
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 25
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 21
- 229920006395 saturated elastomer Polymers 0.000 claims description 21
- MDXGYYOJGPFFJL-QMMMGPOBSA-N N(alpha)-t-butoxycarbonyl-L-leucine Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)OC(C)(C)C MDXGYYOJGPFFJL-QMMMGPOBSA-N 0.000 claims description 17
- 239000002253 acid Substances 0.000 claims description 15
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 claims description 14
- 229940126543 compound 14 Drugs 0.000 claims description 14
- 229960001904 epirubicin Drugs 0.000 claims description 13
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 13
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 claims description 12
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 claims description 12
- 229910052760 oxygen Inorganic materials 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 10
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 claims description 8
- KQZLRWGGWXJPOS-NLFPWZOASA-N 1-[(1R)-1-(2,4-dichlorophenyl)ethyl]-6-[(4S,5R)-4-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-5-methylcyclohexen-1-yl]pyrazolo[3,4-b]pyrazine-3-carbonitrile Chemical compound ClC1=C(C=CC(=C1)Cl)[C@@H](C)N1N=C(C=2C1=NC(=CN=2)C1=CC[C@@H]([C@@H](C1)C)N1[C@@H](CCC1)CO)C#N KQZLRWGGWXJPOS-NLFPWZOASA-N 0.000 claims description 8
- WZZBNLYBHUDSHF-DHLKQENFSA-N 1-[(3s,4s)-4-[8-(2-chloro-4-pyrimidin-2-yloxyphenyl)-7-fluoro-2-methylimidazo[4,5-c]quinolin-1-yl]-3-fluoropiperidin-1-yl]-2-hydroxyethanone Chemical compound CC1=NC2=CN=C3C=C(F)C(C=4C(=CC(OC=5N=CC=CN=5)=CC=4)Cl)=CC3=C2N1[C@H]1CCN(C(=O)CO)C[C@@H]1F WZZBNLYBHUDSHF-DHLKQENFSA-N 0.000 claims description 8
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 claims description 8
- LJOOWESTVASNOG-UFJKPHDISA-N [(1s,3r,4ar,7s,8s,8as)-3-hydroxy-8-[2-[(4r)-4-hydroxy-6-oxooxan-2-yl]ethyl]-7-methyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl] (2s)-2-methylbutanoate Chemical compound C([C@H]1[C@@H](C)C=C[C@H]2C[C@@H](O)C[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)CC1C[C@@H](O)CC(=O)O1 LJOOWESTVASNOG-UFJKPHDISA-N 0.000 claims description 8
- 229940125773 compound 10 Drugs 0.000 claims description 8
- 229940125797 compound 12 Drugs 0.000 claims description 8
- 229940125758 compound 15 Drugs 0.000 claims description 8
- 229940127204 compound 29 Drugs 0.000 claims description 8
- 229940125877 compound 31 Drugs 0.000 claims description 8
- 229940125898 compound 5 Drugs 0.000 claims description 8
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 claims description 8
- XRWSZZJLZRKHHD-WVWIJVSJSA-N asunaprevir Chemical compound O=C([C@@H]1C[C@H](CN1C(=O)[C@@H](NC(=O)OC(C)(C)C)C(C)(C)C)OC1=NC=C(C2=CC=C(Cl)C=C21)OC)N[C@]1(C(=O)NS(=O)(=O)C2CC2)C[C@H]1C=C XRWSZZJLZRKHHD-WVWIJVSJSA-N 0.000 claims description 7
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 7
- 229940125961 compound 24 Drugs 0.000 claims description 7
- 125000006239 protecting group Chemical group 0.000 claims description 7
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 claims description 6
- YSUIQYOGTINQIN-UZFYAQMZSA-N 2-amino-9-[(1S,6R,8R,9S,10R,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-difluoro-3,12-dihydroxy-3,12-bis(sulfanylidene)-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one Chemical compound NC1=NC2=C(N=CN2[C@@H]2O[C@@H]3COP(S)(=O)O[C@@H]4[C@@H](COP(S)(=O)O[C@@H]2[C@@H]3F)O[C@H]([C@H]4F)N2C=NC3=C2N=CN=C3N)C(=O)N1 YSUIQYOGTINQIN-UZFYAQMZSA-N 0.000 claims description 6
- TVTJUIAKQFIXCE-HUKYDQBMSA-N 2-amino-9-[(2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-7-prop-2-ynyl-1H-purine-6,8-dione Chemical compound NC=1NC(C=2N(C(N(C=2N=1)[C@@H]1O[C@@H]([C@H]([C@H]1O)F)CO)=O)CC#C)=O TVTJUIAKQFIXCE-HUKYDQBMSA-N 0.000 claims description 6
- QBWKPGNFQQJGFY-QLFBSQMISA-N 3-[(1r)-1-[(2r,6s)-2,6-dimethylmorpholin-4-yl]ethyl]-n-[6-methyl-3-(1h-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-yl]-1,2-thiazol-5-amine Chemical compound N1([C@H](C)C2=NSC(NC=3C4=NC=C(N4C=C(C)N=3)C3=CNN=C3)=C2)C[C@H](C)O[C@H](C)C1 QBWKPGNFQQJGFY-QLFBSQMISA-N 0.000 claims description 6
- 229940126639 Compound 33 Drugs 0.000 claims description 6
- OPFJDXRVMFKJJO-ZHHKINOHSA-N N-{[3-(2-benzamido-4-methyl-1,3-thiazol-5-yl)-pyrazol-5-yl]carbonyl}-G-dR-G-dD-dD-dD-NH2 Chemical compound S1C(C=2NN=C(C=2)C(=O)NCC(=O)N[C@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(N)=O)=C(C)N=C1NC(=O)C1=CC=CC=C1 OPFJDXRVMFKJJO-ZHHKINOHSA-N 0.000 claims description 6
- PNUZDKCDAWUEGK-CYZMBNFOSA-N Sitafloxacin Chemical compound C([C@H]1N)N(C=2C(=C3C(C(C(C(O)=O)=CN3[C@H]3[C@H](C3)F)=O)=CC=2F)Cl)CC11CC1 PNUZDKCDAWUEGK-CYZMBNFOSA-N 0.000 claims description 6
- 229940125782 compound 2 Drugs 0.000 claims description 6
- 229940125810 compound 20 Drugs 0.000 claims description 6
- 229940126086 compound 21 Drugs 0.000 claims description 6
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- 229940125851 compound 27 Drugs 0.000 claims description 6
- 238000006482 condensation reaction Methods 0.000 claims description 6
- JAXFJECJQZDFJS-XHEPKHHKSA-N gtpl8555 Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)N[C@H](B1O[C@@]2(C)[C@H]3C[C@H](C3(C)C)C[C@H]2O1)CCC1=CC=C(F)C=C1 JAXFJECJQZDFJS-XHEPKHHKSA-N 0.000 claims description 6
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 claims description 5
- NPRYCHLHHVWLQZ-TURQNECASA-N 2-amino-9-[(2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-7-prop-2-ynylpurin-8-one Chemical compound NC1=NC=C2N(C(N(C2=N1)[C@@H]1O[C@@H]([C@H]([C@H]1O)F)CO)=O)CC#C NPRYCHLHHVWLQZ-TURQNECASA-N 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 5
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 claims description 5
- 229940126142 compound 16 Drugs 0.000 claims description 5
- 229960001330 hydroxycarbamide Drugs 0.000 claims description 5
- ABJSOROVZZKJGI-OCYUSGCXSA-N (1r,2r,4r)-2-(4-bromophenyl)-n-[(4-chlorophenyl)-(2-fluoropyridin-4-yl)methyl]-4-morpholin-4-ylcyclohexane-1-carboxamide Chemical compound C1=NC(F)=CC(C(NC(=O)[C@H]2[C@@H](C[C@@H](CC2)N2CCOCC2)C=2C=CC(Br)=CC=2)C=2C=CC(Cl)=CC=2)=C1 ABJSOROVZZKJGI-OCYUSGCXSA-N 0.000 claims description 4
- STBLNCCBQMHSRC-BATDWUPUSA-N (2s)-n-[(3s,4s)-5-acetyl-7-cyano-4-methyl-1-[(2-methylnaphthalen-1-yl)methyl]-2-oxo-3,4-dihydro-1,5-benzodiazepin-3-yl]-2-(methylamino)propanamide Chemical compound O=C1[C@@H](NC(=O)[C@H](C)NC)[C@H](C)N(C(C)=O)C2=CC(C#N)=CC=C2N1CC1=C(C)C=CC2=CC=CC=C12 STBLNCCBQMHSRC-BATDWUPUSA-N 0.000 claims description 4
- XBNGYFFABRKICK-UHFFFAOYSA-N 2,3,4,5,6-pentafluorophenol Chemical compound OC1=C(F)C(F)=C(F)C(F)=C1F XBNGYFFABRKICK-UHFFFAOYSA-N 0.000 claims description 4
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- USPFMEKVPDBMCG-LBPRGKRZSA-N N-benzyloxycarbonyl-L-leucine Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 USPFMEKVPDBMCG-LBPRGKRZSA-N 0.000 claims description 4
- 150000001299 aldehydes Chemical class 0.000 claims description 4
- 229940044683 chemotherapy drug Drugs 0.000 claims description 4
- 229940125878 compound 36 Drugs 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachloro-phenol Natural products OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 claims description 4
- 230000002829 reductive effect Effects 0.000 claims description 4
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 4
- UAOUIVVJBYDFKD-XKCDOFEDSA-N (1R,9R,10S,11R,12R,15S,18S,21R)-10,11,21-trihydroxy-8,8-dimethyl-14-methylidene-4-(prop-2-enylamino)-20-oxa-5-thia-3-azahexacyclo[9.7.2.112,15.01,9.02,6.012,18]henicosa-2(6),3-dien-13-one Chemical compound C([C@@H]1[C@@H](O)[C@@]23C(C1=C)=O)C[C@H]2[C@]12C(N=C(NCC=C)S4)=C4CC(C)(C)[C@H]1[C@H](O)[C@]3(O)OC2 UAOUIVVJBYDFKD-XKCDOFEDSA-N 0.000 claims description 3
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- MVEAAGBEUOMFRX-UHFFFAOYSA-N ethyl acetate;hydrochloride Chemical compound Cl.CCOC(C)=O MVEAAGBEUOMFRX-UHFFFAOYSA-N 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
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- 239000000243 solution Substances 0.000 description 66
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Definitions
- the present invention relates to pharmaceutical chemistry field, particularly relates to a multi-targeted Ubenimex prodrug derivative and preparation method thereof, and its medical use especially as anti-tumor drug (for solid tumor in particular).
- Aminopeptidase N is a zinc-dependent type II trans-membrane metallopeptidase, belonging to the Gluzincins subfamily of the M1 family, which binds to cellular membrane in a form of a homodimer ( Nucleic Acids Res.,1999, 27(1):325-331 ).
- CD 13 is expressed on diverse cell surfaces, such cells as central nervous system synapse cells, synovial fluid fibroblasts, activated endothelial cells, liver cells, intestinal epithelial cells, placenta, bone marrow progenitors, monocytes, osteoclasts and so on, particularly is enriched on kidney and intestinal brush border ( Haema., 2003, 4(6):453-461 ). Moreover, compared with normal cells, many tumor cells have a relatively high expression of CD 13 on their cell surface, such as melanoma, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer and so on.
- CD 13 is a multifunctional protein, playing a role as proteolytic enzyme, virus receptor, signal transduction molecule on cell surface and so on, while CD 13 is involved in cancer invasion, metastasis, and angiogenesis ( Current Medicinal Chemistry, 2007, 14, 639-647 ). Recently, it is reported that the CD13 on surface of tumor cells could change surrounding tissue microenvironment, and has a significant effect on angiogenesis ( PNAS 2012, 109, 1637-1642 ). In addition, Haraguchi etc.
- CD 13 is a functional bio-marker of semi-dormant liver cancer stem cells (HCSCs) in Human hepatoma cells and its clinical samples, and could prevent an increasing of Reactive Oxygen Species inducing by radiotherapy / chemotherapy to increase resistance for treatment ( J Clin Invest 2010, 120, 3326-3339 ). It is well-known that tumor stem cells are the main factor causing tumor chemotherapy resistance, recurrence and metastasis. It is shown in further studies that, CD13 + liver cancer cells proliferate slowly, and have a capability of self-renewing, differentiating and resisting to treatment, and are related to tumor resistance.
- HCSCs semi-dormant liver cancer stem cells
- CD 13 inhibitors and CD 13 neutralizing antibody can both induce cell apoptosis and affect tumor cells with multi-drug resistance, cytotoxic anti-tumor drug and CD 13 inhibitor can highly enhance inhibiting and killing of tumor, thus the combination of ubenimex with other cytotoxic anti-tumor drugs can highly enhance therapeutic effect compared with ubenimex, what is more, can prevent recurrence and metastasis of tumor ( J Clin Invest 2010, 120, 3326-3339 ).
- Ubenimex (Ubenimex, Bestatin, Ube) is a compound with a dipeptide structure found from a nutrient solution of Streptomyces olivoreticuli, which was used as immunoenhancement for the treatment of leukaemia in 1987 in Japan. And then it was on market in 1998 in China with a trade name Bestatin. Bestatin is an immunoenhancement with targeted anti-cancer capacity and dual mechanism. Lots of studies have reported an inhibiting activity of Bestatin on CD13 with IC 50 value of 2.5-16.9 ⁇ M against CD13.
- Bestatin can inhibit an invasion of a murine melanoma B16BL6 with high metastasis; and also can inhibit a tubular structure formation of HUVECs ( Cancer let, 2004, 216(1):35-42 ). Moreover, mice xenografts experiments suggest that Bestatin can inhibit carcinoma metastasis and angiogenesis induced by tumor cells ( Bio. Pharm. Bull., 1996, 19(1):6-10 ); in clinical study, Bestatin can cooperate with chemotherapy, radiotherapy and combination application to be used in treatment of disease such as leukaemia, multiple myeloma, myelodysplastic syndrome, and other diverse solid tumors. However, mechanism of synergy effect thereof is uncertain.
- 5-Fluorouracil is a kind of pyrimidine fluoride with a chemical name of 2,4-dihydroxy-5-fluoropyrimidine and belongs to antimetabolite anti-tumor drugs, and can inhibit thymidylate synthase, block deoxythymidylate converting into thymidylate, interfere with DNA synthesis and inhibit RNA synthesis in some degree.
- 5-Fluorouracil is a widely used clinical anti-tumor drug for treatment of colon cancer, colorectal cancer, stomach cancer, breast cancer, ovarian cancer, choriocarcinoma, malignant mole, head and neck squamous cell carcinoma, skin cancer, liver cancer, bladder cancer, etc., which presents no cross resistance with other general anti-tumor drugs. Otherwise, many antitumor drugs combining with 5-FU exhibit enhanced cytotoxic effect, there has been some studies on drug combination of 5-FU for treating leukaemia ( Phytomedicine 18 (2011) 362-365 ).
- 5-FU is used widely in clinical and has therapeutic effect on many tumor diseases, while having many disadvantages: the effective dose of 5-FU is less different with its toxic dose, which induces the relatively severe myelosuppression and gastrointestinal toxicity in clinical. Short half-life, poor metabolic stability (degraded to FUH 2 by dihydropyrimidine dehydrogenase soon) and some tumors with high expression of thymidylate synthase resisting to it are also its disadvantages. Because of oral irregular, large individual differences, low fat-solubility and poor tissue penetration, it is difficult to prepare a convenient oral formation of 5-fluorouracil, therefore, it is administrated by artery or vein mostly, and is not suitable for the long-term chemotherapy.
- Xeloda is one of those approved by FDA to be an oral fluoropyrimidine drug with the best clinical effect in present, which could meet or exceed the efficacy of fluorouracil by continuous intravenous administration; atofluding can be progressively degraded into TFU and 5-Fu in body so as to have continuous effect.
- Xeloda and TFU as the positive control drugs, we evaluate the oral pharmacodynamic effect of a multi-target ubenimex-5-FU binded pro-drug derivative synthesized by our.
- Hydroxyurea (Hydroxycarbamide), a nucleoside diphosphate reductase inhibitor, can block reduction of nucleotides to deoxynucleotides, interfere with biosynthesis of purine and pyrimidine bases, selectively impede DNA biosynthesis but has no impact on RNA and protein biosynthesis.
- a cell cycle-specific drug cells in s-phase are sensitive to it. Now, it is mainly used for the treatment of solid tumors, such as malignant melanoma, stomach cancer, colon cancer, breast cancer, bladder cancer and so on.
- Epirubicin an antibiotic anti-tumor drug, is an isomer of doxorubicin, which can directly embed in DNA base pairs in nucleus to interfere with transcription progress, and then prevent formation of mRNA to inhibit biosynthesis of DNA and RNA. Moreover, many other researchers find that epirubicin can inhibit the topoisomerase II and is a cell cycle nonspecific agent.
- Dasatinib (Dasatinib), explored by the Bristol-Myers Squibb corporation, was approved by FDA in June of 2006 for treatment of imatinib-resistant CML patients or patients with imatinib failure.
- As a kind of oral tyrosine kinase inhibitor it can treat chronic myeloid leukemia by preventing leukemia cell excessively proliferating in CML or Ph + ALL patients through targeting tyrosine kinase inhibition enzyme cascade.
- Dasatinib can inhibit Src kinase, thus can prevent other human tumor cells with no BCR-ABL expression, such as PC-3 (prostate cancer cells, IC 50 is 5 ⁇ 9 nmol/L), MDA-MB-211 (breast cancer cells, IC 50 is 10 ⁇ 12 nmol/L), and WiDr (colorectal cancer cells, IC 50 is 38 ⁇ 52 nmol/L), etc.
- PC-3 prostate cancer cells
- MDA-MB-211 breast cancer cells
- WiDr colonal cancer cells
- IC 50 is 38 ⁇ 52 nmol/L
- Lenalidomide (Lenalidomide / Revlimid), explored by the U.S. Celgene corporation, is a derivative of thalidomide with a chemical name of 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione and is used for treating myelodysplastic syndrome and combining with dexamethasone for treating multiple myeloma.
- Lenalidomide has an impact on multiple intracellular biological pathways. Now, all the phases I, II, III clinical researches have been ended.
- phase I and phase II clinical researches demonstrate that it has inhibitory effect on many tumors such as multiple myeloma, prostate cancer, thyroid cancer, renal cancer, melanoma, liver cancer and chronic lymphocytic leukemia ( Cancer Chemotherapy and Pharmacology, 2012, 1-14 ).
- Multi-targeted drug is an important trend in today's drug research.
- a multi-targeted drug refers to a drug which can simultaneously act on diverse targets associated with one disease to have a synergistic therapeutic effect, and exhibits better therapeutic effects, less side effects by completely regulating multiple targets associated with one disease, and is especially useful for treating various major diseases associated with complicated pathological mechanism and polygene, including leukemia and other malignancies, central nervous system diseases, cardiovascular diseases and metabolic disorders and so on. So far, multi-targeted treatment using multi-targeted drug is one of the most effective treatments for therapy of various malignancies including leukemia.
- a mutual pro-drug or a multi-targeted drug has single physical and chemical property and uniform pharmacokinetic characteristics when comparing with drug combination and a multi-targeted drug may avoid problems such as interaction of different components and differential absorption, distribution and metabolism of different components in a drug mixture, and thus can have synergistic effect, especially in a case of low concentration.
- a multi-targeted drug shows an obviously better therapeutic effect than combination of corresponding single-targeted drugs. Therefore, it is of great significance to design and synthesize a novel anti-cancer drug with synergistic therapeutic effect based on hybridization principle to be used to treat tumor recurrence, metastasis and drug resistance, especially binding ubenimex as a tumor stem cells inhibitor with other anti-tumor drugs.
- the present invention is aimed at providing a multi-targeted ubenimex pro-drug derivative and preparation method thereof, and its medical use to overcome the defects in the prior art, and in the present invention, a series of novel mutual pro-drugs are designed and synthesized by blending a CD 13 inhibitor ubenimex into molecular structure of another marketed drug (such as 5-FU, hydroxyurea, epirubicin, dasatinib, and paclitaxel) through an ester bond or an amido bond by using pharmacophore hybridization method.
- a CD 13 inhibitor ubenimex into molecular structure of another marketed drug (such as 5-FU, hydroxyurea, epirubicin, dasatinib, and paclitaxel) through an ester bond or an amido bond by using pharmacophore hybridization method.
- These pro-drugs retain an inhibition activity against CD 13, which will play a targeted anti-cancer role on inhibiting tumor stem cells by inhibiting CD 13 after these CD 13 inhibitors entering into body.
- R in general structural formula (1) represents one of the following substituents: in the substituents above, n represents 1 ⁇ 6;
- X represents NH or O;
- R 1 represents H, CH 3 or CH 2 CH 3 ;
- R 2 represents a L - amino acid residue side chain, which is at least one of the following groups: H, CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH 3 CH 2 CH(CH 3 ), CH 2 OH, CH 3 CH(OH), CH 3 SCH 2 CH 2 , CH 2 Ph, OH- p -PhCH 2 ;
- R 3 represents:
- R 4 represents:
- R may represent the following substituents: wherein, in the substituents above, n represents 1 ⁇ 6; X represents NH or O; R 2 represents a L - amino acid residue side chain, which is at least one of the following groups: H, CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH 3 CH 2 CH(CH 3 ), CH 2 OH, CH 3 CH(OH), CH 3 SCH 2 CH 2 , CH 2 Ph, OH- p -PhCH 2 ; R 3 represents: R 4 represents:
- R may represent the following substituents: in the substituents above, n, X, R 1 , R 2 , R 3 are defined as above mentioned, R 3 represents: wherein, the structure of R is preferred to be one of the structures below: in the substituents above, n represents 1 ⁇ 4; X represents NH or O; R 1 represents H; R 3 is especially preferred to represent: R is especially preferred to represent: in the substituents above, n represents 1 or 2; X represents NH or O; R 1 represents H; R 3 represents:
- R represents: in the substituents above , n represents 1; X represents NH; R 1 represents H;
- R may represent the following substituent:
- R may represent the following substituent:
- R may represent the following substituents: wherein, the structure of preferred R is:
- the especially preferred R is:
- the most preferred R is:
- R may represent the following substituents: in the substituents above, n represents 1 ⁇ 6; R 4 represents: wherein, the structure of preferred R is: in the substituents above, n represents 1 ⁇ 6; R 4 represents:
- R 4 in the substituents above, R 4 represents:
- R 4 represents:
- the term "pharmaceutically acceptable salt” is intended to mean a compound represented by formula (I) in salt form that retaining therapeutic effect and nontoxicity. It can be formed to be a cation salt by any alkaline group (such as amino). It is well known that many of the salts are cation salts formed by any alkaline groups (such as amino) in the art. Some salt of those are known to the art.
- the cation salt can be formed by reaction of a corresponding acid with alkali (I), examples of such acids include inorganic acids, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, 2-hydroxy propionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1,2,3-tricarballylic acid, methyl sulfonic acid, ethyl sulfonic acid, benzene sulfonic acid, 4-methyl benzene sulfonic acid, cyclohexyl sulfinic acids, 2-hydroxy benzoic acid, 4-amino-2- hydroxy benzoic acid, and so on.
- inorganic acids for example, hydrochloric acid, sulfuric acid, n
- salts are well known by the skilled person in the art, all the salts in the prior art can be prepared by the skilled person in the art. In addition, the skilled person can take some salts instead of other salts based on factors, such as solubility, stability, easy preparation, etc.. The determination and optimization of the salts are within experiences of the skilled person.
- THF represents tetrahydrofuran
- DCC represents dicyclohexylcarbodiimide
- DMAP represents 4-dimethylaminopyridine
- HCl-AcOE represents ethyl acetate solution saturated by hydrochloric acid
- DCM represents dichloromethane
- EDCI represents 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
- HOBt represents 1-hydroxybenzotriazole
- PPTS represents pyridinium 4-toluenesulfonate
- DHP represents 3,4-dihydro-2 H -pyran
- Pd/C represents palladium carbon hydrogen
- (Boc) 2 O represents di-tert-butyl dicarbonate.
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: 5-fluorouracil is reacted with an aldehyde 1-1 to provide intermediate 2', intermediate 2' is reacted with Boc- L -leucine via condensation reaction to give intermediate 3', protecting group of intermediate 3' is removed to provide compound 4', compound 4' is reacted with Boc-AHPA via condensation reaction to give compound 5', compound 5' is deprotected to give target compound 6'; the specific synthesis route thereof is as follows: wherein, R 1 represents: H, CH 3 , or CH 2 CH 3 .
- the synthesis process of BC-01 includes: 5-fluorouracil is reacted with 37% formaldehyde solution to provide intermediate 2, intermediate 2 is reacted with Boc- L -leucine via condensation reaction to give intermediate 3, protecting group of intermediate 3 is removed to provide compound 4, compound 4 is reacted with Boc-AHPA via condensation reaction to give compound 5, compound 5 is deprotected to give target compound 6 (BC-01); the specific synthesis route thereof is as follows:
- 5-fluorouracil is dissolved in 37% formaldehyde solution to have a reaction at room temperature to provide intermediate 2
- intermediate 2 dissolved in anhydrous THF is reacted with Boc- L -leucine in presence of catalyst DCC and DMAP to give intermediate 3
- protecting group of intermediate 3 is removed in HCl-AcOE solution to provide compound 4
- compound 4 is condensed with Boc-AHPA via EDCl and HOBt in anhydrous DCM to give compound 5
- compound 5 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 6 (BC-01).
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: 5-fluorouracil is reacted with an aldehyde 1-1 to provide intermediate 2'; the hydroxyl group of compound 7 is protected by DHP to give intermediate 8, the benzyl group of intermediate 8 is removed to provide compound 9, compound 9 is condensed with intermediate 2' to give compound 10', compound 10' is deprotected via acid to provide target compound 6'; the specific synthesis route thereof is as follows: wherein, R 1 represents: H, CH 3 , or CH 2 CH 3 , DHP represents 3,4-dihydro-2H-pyran.
- the synthesis process of BC-01 includes: 5-fluorouracil is reacted with 37% formaldehyde solution to provide intermediate 2, the hydroxyl group of compound 7 is protected by DHP to give intermediate 8, the benzyl group of intermediate 8 is removed to provide compound 9, compound 9 is condensed with intermediate 2 to give compound 10, compound 10 is deprotected via acid to provide target compound 6 (BC-01); the specific synthesis route thereof is as follows:
- 5-fluorouracil is dissolved in 37% formaldehyde solution to have a reaction at room temperature to provide intermediate 2, the hydroxyl group of compound 7 dissolved in anhydrous DCM is protected to give intermediate 8 by reacting with DHP and PPTS, the benzyl group of intermediate 8 is removed to provide compound 9 by being treated with Pd/C in H 2 , compound 9 is condensed with intermediate 2 via EDCI and HOBt in anhydrous DCM to give compound 10, compound 10 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 6 (BC-01).
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: compound 14' or its cationic salt is condensed with compound 9 via EDCI and HOBt in anhydrous DCM to give compound 15', compound 15' is deprotected via acid to give target compound 16'; the specific synthesis route thereof is as follows: wherein, compound 9 represents wherein, compound 14' represents alternatively in compound 14', Y represents O or NH, M represents: wherein, n, R 1 , R 2 and R 3 are defined as above.
- Preferred cationic salt is hydrochloride.
- the synthesis process of BC-02 includes: L - glycine is protected via (Boc) 2 O to give the compound 12, compound 12 is condensed with compound 2 to give compound 13, compound 13 is deprotected via acid to give target compound 14, compound 14 is condensed with compound 9 to give compound 15, compound 15 is deprotected via acid to give target compound 16 (BC-02).
- the specific synthesis route thereof is as follows:
- L - glycine is protected via (Boc) 2 O to give the compound 12
- compound 12 is condensed with compound 2 via EDCl and HOBt in anhydrous DCM to give compound 13
- compound 13 is deprotected via ethyl acetate saturated by hydrochloric acid to give compound 14
- compound 14 is condensed with compound 9 via EDCl and HOBt in anhydrous DCM to give compound 15
- compound 15 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 16 (BC-02).
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: Boc- L -Leucine 17 is condensed with pentafluorophenol to give intermediate 18, intermediate 18 is reacted with hydroxyurea to provide compound 19, protecting group of compound 19 is removed via acid to give compound 20, compound 20 is condensed with Boc-AHPA to give compound 21, compound 21 is deprotected to provide target compound 22; the specific synthesis route thereof is as follows:
- Boc- L -Leucine dissolved in anhydrous THF is reacted with pentafluorophenol in presence of EDCI to give intermediate 18, compound 19 was provided from compound 18 in presence of N-methylmorpholine, protecting group of compound 19 is removed in ethyl acetate solution saturated by hydrochloric acid to give compound 20, compound 20 is condensed with via EDCI and HOBt in anhydrous DCM to give compound 21, compound 21 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 22.
- the preparation method of the multi-targeted Ubenimex pro-drug derivative according to the present invention includes: Epirubicin is protected via (Boc) 2 O to give compound 24, compound 24 is condensed with Cbz- L -leucine to give compound 25, an amino-protecting group of compound 25 is removed to give compound 26, compound 26 is condensed with Boc-AHPA to give compound 27, compound 27 is deprotected to give target compound 28; the specific synthesis route thereof is as follows:
- Epirubicin is protected via (Boc) 2 O to give compound 24, compound 24 is condensed with Cbz- L -leucine via EDCI and HOBt in anhydrous DCM to give compound 25, the amino-protecting group of compound 25 is removed by palladium carbon hydrogen to give compound 26, compound 26 is condensed with Boc-AHPA via EDCI and HOBt in anhydrous DCM to give compound 27, compound 27 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 28.
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: Boc- L -leucine 17 is condensed with compound A-E-H to give compound 29', compound 29' is deprotected via acid to give compound 30', compound 30' is condensed with Boc-AHPA to give compound 31', compound 31' is deprotected to give target compound 32'; the specific synthesis route thereof is as follows: wherein, A-E-H represents or E represents NH or O, A represents:
- the synthesis process of dasatinib derivative 32 includes: Boc- L -leucine is condensed with dasatinib to give compound 29, compound 29 is deprotected via acid to give compound 30, compound 30 is condensed with Boc-AHPA to give compound 31, compound 31 is deprotected to give target compound 32; the specific synthesis route thereof is as follows:
- Boc- L -leucine is condensed with dasatinib via EDCl and HOBt in anhydrous DCM to give compound 29
- compound 29 is deprotected via ethyl acetate saturated by hydrochloric acid to give compound 30
- compound 30 is condensed with Boc-AHPA via EDCl and HOBt in anhydrous DCM to give compound 31
- compound 31 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 32.
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: compound 9 is condensed with Q-Y-H to give target compound 33'; wherein, in Q-Y-H, Y represents NH or O, Q represents:
- the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: compound 9 is condensed with p-hydroxybenzaldehyde to give compound 33, compound 33 is reduced to give alcohol 34, alcohol 34 is reacted with triphosgene to give compound 35, compound 35 is reacted with H-R 4 to form compound 36, compound 36 is deprotected to give target compound 37; the specific synthesis route thereof is as follows:
- the drugs and simple derivatives thereof, for binding with ubenimex can be prepared by method known in the art.
- the multi-targeted ubenimex pro-drug derivative in the present invention can be used to treat or prevent a variety of tumors, especially be used as a drug for solid tumors resistant to chemotherapy drugs, and especially be used as anti-malignant tumor (especially the solid tumors) drugs in medical field.
- the multi-targeted ubenimex pro-drug derivative in the present invention can be used to treat or prevent a variety of tumors.
- the tumors represent solid tumors resistant to chemotherapy drugs preferably.
- the present invention has the structural framework of ubenimex be modified but retaining its group which can chelate with zinc iron so that to find out a novel anti-tumor drug.
- ubenimex be modified but retaining its group which can chelate with zinc iron so that to find out a novel anti-tumor drug.
- We designed and synthesized a series of novel mutual pro-drugs by blending a CD 13 inhibitor ubenimex into the molecular structure of another marketed drug (such as 5-FU, hydroxyurea, epirubicin, dasatinib, and paclitaxel) through an ester bond or an amido bond by using pharmacophore hybridization method.
- These pro-drugs retain an inhibition activity against CD 13, which will play a targeted anti-cancer role on inhibiting tumor stem cells by inhibiting CD 13 after these CD 13 inhibitors entering into body.
- pro-drugs would be metabolized in presence of esterase catalyst in vivo to release ubenimex and another anti-cancer durg fragment, and ubenimex keeps targeting CD 13 to inhibit stem cells, on the other hand, another released anti-cancer drug fragment performs its special pharmacodynamic effect, so as to realize a synergistic anti-cancer effect of two drugs, then to improve anti-tumor activity effectively.
- all the mutual pro-drugs designed by us are salts which can improve water solubility, and be suitable to oral and intravenous administration, while can improve the bioavailability of the two drugs.
- the anti-cancer drugs (such as 5-FU, hydroxyurea, epirubicin, dasatinib, paclitaxel, gemcitabine, and hydroxycamptothecine) we selected are broad-spectrum anti-cancer drugs wildly used in clinic and have a good therapeutic effect on treating liver cancer. In a broad sense, therefore, we expect the mutual drug can prolong residence time of its two drugs in vivo, and improve its pharmacokinetic properties and its bioavailability.
- the pro-drugs in the present invention could improve anti-tumor activity by the synergistic effect between ubenimex, which can inhibit tumor stem cells and angiogenesis of tumor microenvironment, and other anti-cancer drugs such as 5-FU with cytotoxicity, especially have anti-recurrence and anti-metastasis effect by inhibiting tumor stem cells.
- the mixture was reacted for 5h after the ice bath was removed.
- the reactant solution was washed with 10% citric acid, saturated NaHCO 3 and saturated NaCl for 3 times respectively, then dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to achieve a yellowish white solid 7 (4.8 g, 47.5%).
- the reactant solution was washed with 10% citric acid, saturated NaHCO 3 and saturated NaCl for three times, and dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get yellow oil, and separated the oil by using flash column chromatography to obtain colorless oil 13 (1.3 g, 67%).
- Boc-epirubicin (24) 23 (1.09 g, 2 mmol) was dissolved in anhydrous dichloromethane, followed by addition of 0.84 mL of trimethylamine and (Boc) 2 O (0.52 g, 2.4 mmol) in dichloromethane solution was added into the mixture dropwise, the mixture was reacted overnight at room temperature until TLC test gave the reaction was accomplished. After the reaction was accomplished, the reactant solution was washed with 10% citric acid and saturated NaCl for 3 times, dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get compound 24 (1.09 g, 85%).
- the air in the reactant bottle was removed out and was filled with hydrogen by a hydrogen balloon, after being reacted for 12h, filtered the mixture with two layers of filter papers. The solvent was evaporated until the residue was dried to get colorless foam like solid 26 (0.6g, 93.0%).
- Aminopeptidase N and L -Leu- p -nitroanilide as substrate were purchased from Sigma cooperation.
- buffer solution 12.89 g Na 2 HPO 4 •12H 2 O and 2.18 g NaH 2 PO 4 •2H 2 O were dissolved in a 1000 mL volumetric flask, then the mixture was diluted to 1000 mL with fresh distilled water to obtain 50 mM Phosphate Buffer solution (PBS, pH 7.2), which was kept at room temperature to spare.
- PBS Phosphate Buffer solution
- Aminopeptidase N was dissolved in the buffer solution to obtain 0.1IU/mL solution.
- the substrate was dissolved in DMSO to obtain a 16 mmol/mL solution, which was kept in refrigerator to spare.
- Inhibitory ratio absorbance of 100 % - absorbance of compound absorbance of 100 % - absorbance of blank group ⁇ 100 %
- the IC 50 value was calculated by using a fitting curve of Origin 7.5 software.
- the target compounds BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, 22, 28 and 32 represented by formula (I) of the present invention all established an inhibitory activity against aminopeptidase N, wherein Compounds BC-02, BC-03, BC-04, BC-05, BC-06 and BC-07 were much more potent than the positive control drug ubenimex, especially, BC-07 showed great activity with 0.15 ⁇ M.
- Compounds BC-01, 22, 28 and 32 showed a comparable activity with ubenimex. This indicated that synergetic fragment linked at carboxyl group of ubenimex may increase its activity against enzyme without destroying inhibition activity against enzyme of the whole structure.
- Human leukemia cell line K562, clear-cell ovarian carcinoma cell line ES-2, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, cell line Hela, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO were transferred into a culture flask and were cultured in a medium at 37°C in a 5% CO 2 and saturation humidity.
- One bottle of cells in logarithmic growth phase was picked out and was blew and beaten to be uniform with a pipette, then took some cell suspension solution to prepare blood counting plate smear to count cells under inverted microscope, and then adjusted the cell number to be 1 ⁇ 10 5 /mL by adding some culture medium.
- Cells were seeded on a 96-well plate on which also drug tests performed.
- Blank control group, negative control group, positive control group and drug test group were established on the plate while the surrounding wells of the plate were not used (but filled with sterile PBS), wherein the blank control groups were prepared by addition of culture medium 150 ⁇ L per well, the negative control groups were prepared by addition of cell suspension solution 100 ⁇ L per well and culture medium 50 ⁇ L per well, the positive control groups were prepared by addition of cell suspension solution 100 ⁇ L per well and positive control drug solution 50 ⁇ L per well, the drug test groups were prepared by addition of cell suspension solution 100 ⁇ L per well and test compound solution 50 ⁇ L per well, and there were 5 different concentrations of drug in the positive control group and the drug test group respectively: 0.01, 0.1, 1, 10, 100 ⁇ mol ⁇ L -1 , each concentration was established with 3 parallel wells.
- the 96-well plate was incubated for 48 h at 37°C in a CO 2 incubator of 5% CO 2 and saturation humidity. Upon completion of the incubation, 20 ⁇ L MTT solution (concentration was 5mg/mL) was added to each well and incubated for an additional 4h, and after the 96-well plate was centrifuged at 2000 rpm for 30 min, medium of each well was suck out carefully and removed and 100 ⁇ L DMSO was added instead, followed by vibrating on a tablet shaker for 15min to re-dissolve formazan crystals completely.
- MTT solution concentration was 5mg/mL
- BC-01, BC-02, 22, 28 and 32 represented by formula (I) of the present invention established an obvious anti-proliferation effect against all the tumor cells above.
- BC-01 showed much higher proliferation inhibition activity against cell line Hela, and had an obvious proliferation inhibition activity against clear-cell ovarian carcinoma cell line ES-2, human leukemia cell line K562, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO as well; BC-02 established a great proliferation inhibition activity against clear-cell ovarian carcinoma ES-2 cell line.
- Compounds 22, 28 and 32 were much more potent than 5-FU on proliferation inhibition activity.
- the H22 tumor bearing mice were randomized to 4 groups (10 mice per group) after being weighted.
- the size of tumor was measured by using a vernier calliper and weight of mouse by using an electronic balance at the beginning and ending of every cycle, then calculated their mean value.
- the mice were sacrificed after 13 days and the tumors were taken out and weighted.
- the H22 tumor-bearing mice were randomized to 4 groups (7 mice per group) after being weighted.
- the ascites was extracted from H22 tumour bearing mice with well growth, followed by adding sterile PBS to dilute the ascites to a concentration of 8.5 ⁇ 10 7 cells/mL. 100uL of the cells solution was suck up by a 1mL sterile injector to inoculate into right oxter of mice. After 3 days, the mice were weighted and randomized to several groups (almost 8 mice per group, and a bit more in control group). These mice were administrated a predetermined dosage by intravenous or oral gavage with two days off when having administrated for 5 days until two cycles one of which had 7 days. The body weight of mouse was recorded at the beginning and ending of every cycle,
- mice After being administrated for two cycles, the mice were weighted and recorded, then sacrificed by cervical dislocation to get lung, liver and spleen weighted respectively.
- inhibitory ratio 100 % mean tumor weight control group - mean tumor weight of drug administrated group / mean tumor weight of control group ⁇ 100.
- compounds BC-01 and BC-02 all showed excellent anti-tumor growth activity, what's more, at a dosage of 0.15mmol/kg/d of intravenous administrated BC-02, no tumor appeared until the end of the experiment.
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Abstract
Description
- The present invention relates to pharmaceutical chemistry field, particularly relates to a multi-targeted Ubenimex prodrug derivative and preparation method thereof, and its medical use especially as anti-tumor drug (for solid tumor in particular).
- Aminopeptidase N (APN/CD13) is a zinc-dependent type II trans-membrane metallopeptidase, belonging to the Gluzincins subfamily of the M1 family, which binds to cellular membrane in a form of a homodimer (Nucleic Acids Res.,1999, 27(1):325-331 ). CD 13 is expressed on diverse cell surfaces, such cells as central nervous system synapse cells, synovial fluid fibroblasts, activated endothelial cells, liver cells, intestinal epithelial cells, placenta, bone marrow progenitors, monocytes, osteoclasts and so on, particularly is enriched on kidney and intestinal brush border ( Haema., 2003, 4(6):453-461). Moreover, compared with normal cells, many tumor cells have a relatively high expression of CD 13 on their cell surface, such as melanoma, ovarian cancer, prostate cancer, colon cancer, pancreatic cancer, breast cancer, lung cancer and so on.
- In recent studies, it is found CD 13 is a multifunctional protein, playing a role as proteolytic enzyme, virus receptor, signal transduction molecule on cell surface and so on, while CD 13 is involved in cancer invasion, metastasis, and angiogenesis (Current Medicinal Chemistry, 2007, 14, 639-647). Recently, it is reported that the CD13 on surface of tumor cells could change surrounding tissue microenvironment, and has a significant effect on angiogenesis (PNAS 2012, 109, 1637-1642). In addition, Haraguchi etc. suggest that CD 13 is a functional bio-marker of semi-dormant liver cancer stem cells (HCSCs) in Human hepatoma cells and its clinical samples, and could prevent an increasing of Reactive Oxygen Species inducing by radiotherapy / chemotherapy to increase resistance for treatment ( J Clin Invest 2010, 120, 3326-3339 ). It is well-known that tumor stem cells are the main factor causing tumor chemotherapy resistance, recurrence and metastasis. It is shown in further studies that, CD13+ liver cancer cells proliferate slowly, and have a capability of self-renewing, differentiating and resisting to treatment, and are related to tumor resistance. CD 13 inhibitors and CD 13 neutralizing antibody can both induce cell apoptosis and affect tumor cells with multi-drug resistance, cytotoxic anti-tumor drug and CD 13 inhibitor can highly enhance inhibiting and killing of tumor, thus the combination of ubenimex with other cytotoxic anti-tumor drugs can highly enhance therapeutic effect compared with ubenimex, what is more, can prevent recurrence and metastasis of tumor ( J Clin Invest 2010, 120, 3326-3339 ).
- Ubenimex (Ubenimex, Bestatin, Ube) is a compound with a dipeptide structure found from a nutrient solution of Streptomyces olivoreticuli, which was used as immunoenhancement for the treatment of leukaemia in 1987 in Japan. And then it was on market in 1998 in China with a trade name Bestatin. Bestatin is an immunoenhancement with targeted anti-cancer capacity and dual mechanism. Lots of studies have reported an inhibiting activity of Bestatin on CD13 with IC50 value of 2.5-16.9µM against CD13. In vitro, Bestatin can inhibit an invasion of a murine melanoma B16BL6 with high metastasis; and also can inhibit a tubular structure formation of HUVECs (Cancer let, 2004, 216(1):35-42 ). Moreover, mice xenografts experiments suggest that Bestatin can inhibit carcinoma metastasis and angiogenesis induced by tumor cells ( Bio. Pharm. Bull., 1996, 19(1):6-10); in clinical study, Bestatin can cooperate with chemotherapy, radiotherapy and combination application to be used in treatment of disease such as leukaemia, multiple myeloma, myelodysplastic syndrome, and other diverse solid tumors. However, mechanism of synergy effect thereof is uncertain.
- Recently, in a mice liver cancer xenograft experiment, the combination of ubenimex and 5-Fluorouracil presented an enhanced effect on inhibiting tumor comparing with a single treatment using ubenimex or 5-Fluorouracil (shown in
Fig1, 2 ). Otherwise, it was found in further experiment that, the liver cells of a mouse treated by 5-Fluorouracil before was transplanted into other mouse, and by comparing the tumor recurrence of a mice group using ubenimex with the mice group without using ubenimex, it results that, tumor did not recur in the mice group using ubenimex, but the mice group without using ubenimex presented tumor recurrence cases. This study provides great evidence for combination of ubenimex with 5-Fluorouracil and other chemotherapeutics ( J Clin Invest 2010, 120, 3326-3339). And it also validates the importance of inhibiting tumor stem cells in tumor chemotherapy. - 5-Fluorouracil (5-Fluorouracil, 5-Fu) is a kind of pyrimidine fluoride with a chemical name of 2,4-dihydroxy-5-fluoropyrimidine and belongs to antimetabolite anti-tumor drugs, and can inhibit thymidylate synthase, block deoxythymidylate converting into thymidylate, interfere with DNA synthesis and inhibit RNA synthesis in some degree. 5-Fluorouracil is a widely used clinical anti-tumor drug for treatment of colon cancer, colorectal cancer, stomach cancer, breast cancer, ovarian cancer, choriocarcinoma, malignant mole, head and neck squamous cell carcinoma, skin cancer, liver cancer, bladder cancer, etc., which presents no cross resistance with other general anti-tumor drugs. Otherwise, many antitumor drugs combining with 5-FU exhibit enhanced cytotoxic effect, there has been some studies on drug combination of 5-FU for treating leukaemia (Phytomedicine 18 (2011) 362-365). However, 5-FU is used widely in clinical and has therapeutic effect on many tumor diseases, while having many disadvantages: the effective dose of 5-FU is less different with its toxic dose, which induces the relatively severe myelosuppression and gastrointestinal toxicity in clinical. Short half-life, poor metabolic stability (degraded to FUH2 by dihydropyrimidine dehydrogenase soon) and some tumors with high expression of thymidylate synthase resisting to it are also its disadvantages. Because of oral irregular, large individual differences, low fat-solubility and poor tissue penetration, it is difficult to prepare a convenient oral formation of 5-fluorouracil, therefore, it is administrated by artery or vein mostly, and is not suitable for the long-term chemotherapy.
- In decades, many researchers want to reduce or avoid the disadvantages of 5-FU. Among which, the main method is developing pro-drug of 5-FU to improve metabolic stability, reduce toxicity and increase therapeutic index of it. So far, many pro-drug derivatives of 5-FU have been synthesized and some of those have been used in clinial, such as tegafur (FTO), carmofur (HCFU), doxifluridine, capecitabine (Xeloda), atofluding (ATFU), BOF-A2 and so on. Xeloda is one of those approved by FDA to be an oral fluoropyrimidine drug with the best clinical effect in present, which could meet or exceed the efficacy of fluorouracil by continuous intravenous administration; atofluding can be progressively degraded into TFU and 5-Fu in body so as to have continuous effect. In this invention, by using Xeloda and TFU as the positive control drugs, we evaluate the oral pharmacodynamic effect of a multi-target ubenimex-5-FU binded pro-drug derivative synthesized by ourselves.
- Hydroxyurea (Hydroxycarbamide), a nucleoside diphosphate reductase inhibitor, can block reduction of nucleotides to deoxynucleotides, interfere with biosynthesis of purine and pyrimidine bases, selectively impede DNA biosynthesis but has no impact on RNA and protein biosynthesis. As a cell cycle-specific drug, cells in s-phase are sensitive to it. Now, it is mainly used for the treatment of solid tumors, such as malignant melanoma, stomach cancer, colon cancer, breast cancer, bladder cancer and so on.
- Epirubicin (Epirubicin), an antibiotic anti-tumor drug, is an isomer of doxorubicin, which can directly embed in DNA base pairs in nucleus to interfere with transcription progress, and then prevent formation of mRNA to inhibit biosynthesis of DNA and RNA. Moreover, many other researchers find that epirubicin can inhibit the topoisomerase II and is a cell cycle nonspecific agent. Now, it is mainly used for the treatment of solid tumors in clinical, such as acute leukemia, nephroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, malignant lymphoma, breast cancer, bronchial carcinoma, ovarian cancer, liver cancer (including primary hepatocellular carcinoma and metastatic hepatocellular carcinoma) and so on. So far, combination application of epirubicin has been widely reported, for example, combination of epirubicin with paclitaxel or sorafenib is useful for treatment of advanced breast cancer.
- Dasatinib (Dasatinib), explored by the Bristol-Myers Squibb corporation, was approved by FDA in June of 2006 for treatment of imatinib-resistant CML patients or patients with imatinib failure. As a kind of oral tyrosine kinase inhibitor, it can treat chronic myeloid leukemia by preventing leukemia cell excessively proliferating in CML or Ph + ALL patients through targeting tyrosine kinase inhibition enzyme cascade. Other than, Dasatinib can inhibit Src kinase, thus can prevent other human tumor cells with no BCR-ABL expression, such as PC-3 (prostate cancer cells, IC50 is 5∼9 nmol/L), MDA-MB-211 (breast cancer cells, IC50 is 10∼12 nmol/L), and WiDr (colorectal cancer cells, IC50 is 38∼52 nmol/L), etc. ( Proc Am Assoc Cancer Res, 2005, 46: 159).
- Lenalidomide (Lenalidomide / Revlimid), explored by the U.S. Celgene corporation, is a derivative of thalidomide with a chemical name of 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione and is used for treating myelodysplastic syndrome and combining with dexamethasone for treating multiple myeloma. Lenalidomide has an impact on multiple intracellular biological pathways. Now, all the phases I, II, III clinical researches have been ended. The studies of phase I and phase II clinical researches demonstrate that it has inhibitory effect on many tumors such as multiple myeloma, prostate cancer, thyroid cancer, renal cancer, melanoma, liver cancer and chronic lymphocytic leukemia ( Cancer Chemotherapy and Pharmacology, 2012, 1-14 ).
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- Multi-targeted drug (multitarget drugs) is an important trend in today's drug research. A multi-targeted drug refers to a drug which can simultaneously act on diverse targets associated with one disease to have a synergistic therapeutic effect, and exhibits better therapeutic effects, less side effects by completely regulating multiple targets associated with one disease, and is especially useful for treating various major diseases associated with complicated pathological mechanism and polygene, including leukemia and other malignancies, central nervous system diseases, cardiovascular diseases and metabolic disorders and so on. So far, multi-targeted treatment using multi-targeted drug is one of the most effective treatments for therapy of various malignancies including leukemia.
- A mutual pro-drug or a multi-targeted drug has single physical and chemical property and uniform pharmacokinetic characteristics when comparing with drug combination and a multi-targeted drug may avoid problems such as interaction of different components and differential absorption, distribution and metabolism of different components in a drug mixture, and thus can have synergistic effect, especially in a case of low concentration. A multi-targeted drug shows an obviously better therapeutic effect than combination of corresponding single-targeted drugs. Therefore, it is of great significance to design and synthesize a novel anti-cancer drug with synergistic therapeutic effect based on hybridization principle to be used to treat tumor recurrence, metastasis and drug resistance, especially binding ubenimex as a tumor stem cells inhibitor with other anti-tumor drugs.
- The present invention is aimed at providing a multi-targeted ubenimex pro-drug derivative and preparation method thereof, and its medical use to overcome the defects in the prior art, and in the present invention, a series of novel mutual pro-drugs are designed and synthesized by blending a CD 13 inhibitor ubenimex into molecular structure of another marketed drug (such as 5-FU, hydroxyurea, epirubicin, dasatinib, and paclitaxel) through an ester bond or an amido bond by using pharmacophore hybridization method. These pro-drugs retain an inhibition activity against CD 13, which will play a targeted anti-cancer role on inhibiting tumor stem cells by inhibiting CD 13 after these CD 13 inhibitors entering into body.
- In order to solve the above problems, a technical solution of the present invention is as follows: to provide a multi-target pro-drug derivatives of ubenimex as represented by general formula (I), as well as an enantiomer, a diastereoisomer and a racemate thereof, and a pharmaceutically acceptable salt or solvate thereof:
R3 represents: - In the general structural formula (I) of the present invention, R may represent the following substituents:
R3 represents: - In the general structural formula (I) of the present invention, R may represent the following substituents:
-
-
-
-
-
-
-
-
-
- In the multi-targeted Ubenimex pro-drug derivative of the present invention, the term "pharmaceutically acceptable salt" is intended to mean a compound represented by formula (I) in salt form that retaining therapeutic effect and nontoxicity. It can be formed to be a cation salt by any alkaline group (such as amino). It is well known that many of the salts are cation salts formed by any alkaline groups (such as amino) in the art. Some salt of those are known to the art. The cation salt can be formed by reaction of a corresponding acid with alkali (I), examples of such acids include inorganic acids, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, 2-hydroxy propionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1,2,3-tricarballylic acid, methyl sulfonic acid, ethyl sulfonic acid, benzene sulfonic acid, 4-methyl benzene sulfonic acid, cyclohexyl sulfinic acids, 2-hydroxy benzoic acid, 4-amino-2- hydroxy benzoic acid, and so on. These salts are well known by the skilled person in the art, all the salts in the prior art can be prepared by the skilled person in the art. In addition, the skilled person can take some salts instead of other salts based on factors, such as solubility, stability, easy preparation, etc.. The determination and optimization of the salts are within experiences of the skilled person.
- In the present invention, THF represents tetrahydrofuran, DCC represents dicyclohexylcarbodiimide, DMAP represents 4-dimethylaminopyridine, HCl-AcOE represents ethyl acetate solution saturated by hydrochloric acid, DCM represents dichloromethane, EDCI represents 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HOBt represents 1-hydroxybenzotriazole, PPTS represents pyridinium 4-toluenesulfonate, DHP represents 3,4-dihydro-2H-pyran, Pd/C, H2 represents palladium carbon hydrogen, (Boc)2O represents di-tert-butyl dicarbonate.
- The preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: 5-fluorouracil is reacted with an aldehyde 1-1 to provide intermediate 2', intermediate 2' is reacted with Boc-L-leucine via condensation reaction to give intermediate 3', protecting group of intermediate 3' is removed to provide compound 4', compound 4' is reacted with Boc-AHPA via condensation reaction to give compound 5', compound 5' is deprotected to give target compound 6'; the specific synthesis route thereof is as follows:
- Specifically, for example, the synthesis process of BC-01 (the skilled in the art can prepare other compound 6' based on the following synthesis process of BC-01) includes: 5-fluorouracil is reacted with 37% formaldehyde solution to provide intermediate 2, intermediate 2 is reacted with Boc-L-leucine via condensation reaction to give intermediate 3, protecting group of intermediate 3 is removed to provide compound 4, compound 4 is reacted with Boc-AHPA via condensation reaction to give
compound 5,compound 5 is deprotected to give target compound 6 (BC-01); the specific synthesis route thereof is as follows: - More specifically, 5-fluorouracil is dissolved in 37% formaldehyde solution to have a reaction at room temperature to provide intermediate 2, intermediate 2 dissolved in anhydrous THF is reacted with Boc-L-leucine in presence of catalyst DCC and DMAP to give intermediate 3, protecting group of intermediate 3 is removed in HCl-AcOE solution to provide compound 4, compound 4 is condensed with Boc-AHPA via EDCl and HOBt in anhydrous DCM to give
compound 5,compound 5 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 6 (BC-01). - Any specific conditions of each reaction in the synthesis route above can be routine reaction conditions known in the art.
- The preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: 5-fluorouracil is reacted with an aldehyde 1-1 to provide intermediate 2'; the hydroxyl group of compound 7 is protected by DHP to give intermediate 8, the benzyl group of intermediate 8 is removed to provide compound 9, compound 9 is condensed with intermediate 2' to give compound 10', compound 10' is deprotected via acid to provide target compound 6'; the specific synthesis route thereof is as follows:
- Specifically, for example, the synthesis process of BC-01 (the skilled in the art can prepare other compound 6' based on the following synthesis process of BC-01) includes: 5-fluorouracil is reacted with 37% formaldehyde solution to provide intermediate 2, the hydroxyl group of compound 7 is protected by DHP to give intermediate 8, the benzyl group of intermediate 8 is removed to provide compound 9, compound 9 is condensed with intermediate 2 to give
compound 10,compound 10 is deprotected via acid to provide target compound 6 (BC-01); the specific synthesis route thereof is as follows: - More specifically, 5-fluorouracil is dissolved in 37% formaldehyde solution to have a reaction at room temperature to provide intermediate 2, the hydroxyl group of compound 7 dissolved in anhydrous DCM is protected to give intermediate 8 by reacting with DHP and PPTS, the benzyl group of intermediate 8 is removed to provide compound 9 by being treated with Pd/C in H2, compound 9 is condensed with intermediate 2 via EDCI and HOBt in anhydrous DCM to give
compound 10,compound 10 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 6 (BC-01). - Any specific conditions of each reaction in the synthesis route above can be routine reaction conditions known in the art.
- The preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: compound 14' or its cationic salt is condensed with compound 9 via EDCI and HOBt in anhydrous DCM to give compound 15', compound 15' is deprotected via acid to give target compound 16'; the specific synthesis route thereof is as follows:
when Y represents NH and M represents - Specifically, for example, the synthesis process of BC-02 (the skilled in the art can prepare other compound 16' based on the following synthesis process of BC-02) includes: L- glycine is protected via (Boc)2O to give the compound 12, compound 12 is condensed with compound 2 to give compound 13, compound 13 is deprotected via acid to give target compound 14, compound 14 is condensed with compound 9 to give compound 15, compound 15 is deprotected via acid to give target compound 16 (BC-02). The specific synthesis route thereof is as follows:
- More specifically, L- glycine is protected via (Boc)2O to give the compound 12, compound 12 is condensed with compound 2 via EDCl and HOBt in anhydrous DCM to give compound 13, compound 13 is deprotected via ethyl acetate saturated by hydrochloric acid to give compound 14, compound 14 is condensed with compound 9 via EDCl and HOBt in anhydrous DCM to give compound 15, compound 15 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 16 (BC-02).
- Any specific conditions of each reaction in the synthesis route above can be routine reaction conditions known in the art.
- The preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: Boc-L-Leucine 17 is condensed with pentafluorophenol to give intermediate 18, intermediate 18 is reacted with hydroxyurea to provide compound 19, protecting group of compound 19 is removed via acid to give
compound 20,compound 20 is condensed with Boc-AHPA to give compound 21, compound 21 is deprotected to provide target compound 22;
the specific synthesis route thereof is as follows: - More specifically, Boc-L-Leucine dissolved in anhydrous THF is reacted with pentafluorophenol in presence of EDCI to give intermediate 18, compound 19 was provided from compound 18 in presence of N-methylmorpholine, protecting group of compound 19 is removed in ethyl acetate solution saturated by hydrochloric acid to give
compound 20,compound 20 is condensed with via EDCI and HOBt in anhydrous DCM to give compound 21, compound 21 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 22. - Any specific conditions of each reaction in the synthesis route above can be routine reaction conditions known in the art.
- The preparation method of the multi-targeted Ubenimex pro-drug derivative according to the present invention includes: Epirubicin is protected via (Boc)2O to give compound 24, compound 24 is condensed with Cbz-L-leucine to give compound 25, an amino-protecting group of compound 25 is removed to give compound 26, compound 26 is condensed with Boc-AHPA to give compound 27, compound 27 is deprotected to give target compound 28; the specific synthesis route thereof is as follows:
- More specifically, Epirubicin is protected via (Boc)2O to give compound 24, compound 24 is condensed with Cbz-L-leucine via EDCI and HOBt in anhydrous DCM to give compound 25, the amino-protecting group of compound 25 is removed by palladium carbon hydrogen to give compound 26, compound 26 is condensed with Boc-AHPA via EDCI and HOBt in anhydrous DCM to give compound 27, compound 27 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 28.
- Any specific conditions of each reaction in the synthesis route above can be routine reaction conditions known in the art.
- The preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: Boc-L-leucine 17 is condensed with compound A-E-H to give compound 29', compound 29' is deprotected via acid to give compound 30', compound 30' is condensed with Boc-AHPA to give compound 31', compound 31' is deprotected to give target compound 32'; the specific synthesis route thereof is as follows:
- Specifically, for example, the synthesis process of dasatinib derivative 32 (the skilled in the art can prepare other compound 32' based on the following synthesis process of dasatinib derivative 32) includes: Boc-L-leucine is condensed with dasatinib to give compound 29, compound 29 is deprotected via acid to give
compound 30,compound 30 is condensed with Boc-AHPA to give compound 31, compound 31 is deprotected to give target compound 32; the specific synthesis route thereof is as follows: - More specifically, Boc-L-leucine is condensed with dasatinib via EDCl and HOBt in anhydrous DCM to give compound 29, compound 29 is deprotected via ethyl acetate saturated by hydrochloric acid to give
compound 30,compound 30 is condensed with Boc-AHPA via EDCl and HOBt in anhydrous DCM to give compound 31, compound 31 is deprotected via ethyl acetate saturated by hydrochloric acid to give target compound 32. - Any specific conditions of each reaction in the synthesis route above can be routine reaction conditions known in the art.
-
- Specifically, the preparation method of the multi-targeted ubenimex pro-drug derivative according to the present invention includes: compound 9 is condensed with p-hydroxybenzaldehyde to give compound 33, compound 33 is reduced to give alcohol 34, alcohol 34 is reacted with triphosgene to give compound 35, compound 35 is reacted with H-R4 to form compound 36, compound 36 is deprotected to give target compound 37; the specific synthesis route thereof is as follows:
- In the present invention, the drugs and simple derivatives thereof, for binding with ubenimex, can be prepared by method known in the art.
- The multi-targeted ubenimex pro-drug derivative in the present invention can be used to treat or prevent a variety of tumors, especially be used as a drug for solid tumors resistant to chemotherapy drugs, and especially be used as anti-malignant tumor (especially the solid tumors) drugs in medical field.
- The multi-targeted ubenimex pro-drug derivative in the present invention can be used to treat or prevent a variety of tumors. Wherein, the tumors represent solid tumors resistant to chemotherapy drugs preferably.
- According to the pro-drug principle and hybridization principle, the present invention has the structural framework of ubenimex be modified but retaining its group which can chelate with zinc iron so that to find out a novel anti-tumor drug. We designed and synthesized a series of novel mutual pro-drugs by blending a CD 13 inhibitor ubenimex into the molecular structure of another marketed drug (such as 5-FU, hydroxyurea, epirubicin, dasatinib, and paclitaxel) through an ester bond or an amido bond by using pharmacophore hybridization method. These pro-drugs retain an inhibition activity against CD 13, which will play a targeted anti-cancer role on inhibiting tumor stem cells by inhibiting CD 13 after these CD 13 inhibitors entering into body. In addition, These pro-drugs would be metabolized in presence of esterase catalyst in vivo to release ubenimex and another anti-cancer durg fragment, and ubenimex keeps targeting CD 13 to inhibit stem cells, on the other hand, another released anti-cancer drug fragment performs its special pharmacodynamic effect, so as to realize a synergistic anti-cancer effect of two drugs, then to improve anti-tumor activity effectively. Furthermore, all the mutual pro-drugs designed by us are salts which can improve water solubility, and be suitable to oral and intravenous administration, while can improve the bioavailability of the two drugs. What's more, the anti-cancer drugs (such as 5-FU, hydroxyurea, epirubicin, dasatinib, paclitaxel, gemcitabine, and hydroxycamptothecine) we selected are broad-spectrum anti-cancer drugs wildly used in clinic and have a good therapeutic effect on treating liver cancer. In a broad sense, therefore, we expect the mutual drug can prolong residence time of its two drugs in vivo, and improve its pharmacokinetic properties and its bioavailability. The pro-drugs in the present invention could improve anti-tumor activity by the synergistic effect between ubenimex, which can inhibit tumor stem cells and angiogenesis of tumor microenvironment, and other anti-cancer drugs such as 5-FU with cytotoxicity, especially have anti-recurrence and anti-metastasis effect by inhibiting tumor stem cells.
-
-
Fig.1 illustrates the tumor size of control mice and combination of ubenimex and 5-FU treated mice. -
Fig.2 illustrates the tumor volume of Kunming mice. -
Fig.3 illustrates the body weight changing curve of Kunming mice by oral administration. -
Fig.4 illustrates the tumor weight of Kunming mice by oral administration. -
Fig.5 illustrates the tumor inhibitory ratio of compounds by oral administration. -
Fig.6 illustrates the photographs of tumors of every groups by oral administration. -
Fig.7 illustrates the body weight changing curve of Kunming mice by intravenous injection. -
Fig.8 illustrates the tumor weight of Kunming mice by intravenous injection. -
Fig.9 illustrates the tumor inhibitory ratio of compounds by intravenous injection. -
Fig.10 illustrates the photographs of tumors of every groups by intravenous injection. -
Fig11 illustrates the tumor weight and its deviation, wherein, *signed groups were treated by oral gavage, the other were treated by tail intravenous injection. -
Fig.12 illustrates the inhibitory ratio of compounds, wherein, *signed groups were treated by oral gavage, the other were treated by tail intravenous injection. -
Fig.13 illustrates animal weight changing curve, wherein, *signed groups were treated by oral gavage, the other were treated by tail intravenous injection. -
Fig.14 illustrates the photographs of tumors obtained in experiments, wherein, *signed groups were treated by oral gavage., the other were treated by intravenous injection, besides, the "O" means no tumor detected, while the "X" means animal dead. - The following examples are served to exemplify the present invention, but not to limit the scope of the invention.
- Preparation of 5-fluoro-1-hydroxymethylpyrimidine-2,4(1H,3H)-dione (2): 5-FU (0.26 g, 2 mmol) was dissolved in 1 mL of 37% formaldehyde solution and the mixture was reacted at 60°C for 2 h under oil bath. After evaporating the solvent under reduced pressure, the residue was dried under vacuum to get colorless viscous oil 2 (0.3 g, yield: 94%).
- Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((tert-butoxycarbonyl)amino)-4-methylpentanoate (3): 2 (0.3 g, 1.9 mmol) was dissolved in acetonitrile, followed by adding Boc-L-leucine (0.7 g, 2.8 mmol), DCC (0.6 g, 2.8 mmol) and DMAP (0.03 g) into it while stirring under ice bath. After the ice bath was removed, the obtained mixture was stirred at room temperature for 12 h. Filtered and evaporated the solvent, extracted with ethyl acetate, the organic phase was washed with water, 1 M citric acid, saturated sodium bicarbonate and saturated sodium chloride solution in turn. Dried with anhydrous sodium sulfate, filtered it and evaporated the solvent to get colorless oil 3 (0.42 g, 60%).
- Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-(amino)-4-methylpentanoate hydrochloride (4): 3 (0.37 g, 1.0 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and the solution was reacted at room temperature for 2h. Filtered the solution then to get white powder 4 (0.26g, 83%).
- Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpenta noate (5): Boc-AHPA (0.3 g, 1.0 mmol) was dissolved in anhydrous dichloromethane, and followed by the addition of EDCI (0.3 g,1.5 mmol) and HOBt (0.2 g, 1.5mmol) under ice bath, after stirring for 0.5 h, 4 (0.3 g, 1 mmol) and 0.2 mL of triethylamine were added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. After the reaction was accomplished, the organic layer was washed with water, 1 M citric acid, saturated sodium bicarbonate and saturated sodium chloride solution respectively, then dried with anhydrous sodium sulfate, filtered it and evaporated the solvent to get white solid 5 (0.33g, 55%).
- Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-(amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate hydrochloride (6(BC-01)): 5 (0.33g, 0.55 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and the solution was reacted at room temperature for 2h. Filtered it to get white solid 6(BC-01) (0.23g, 85%). ESI-MS m/z:451.6 (M+H)+,1H-NMR(600 MHz DMSO): δ 0.84-0.87 (m, 6H), 1.53 (m, 1H), 1.60-1.68 (m, 2H), 2.89-2.95 (m, 2H), 3.99-4.03 (m, 2H), 4.25 (m, 1H), 5.56-5.61 (m, 2H),6.80 (s, 1H), 7.26-7.35 (m, 5H), 8.04 (s, 3H), 8.13(d, J=6.6 Hz, 1H),8.47(d, J=7.2 Hz, 1H), 12.01 (s, 1H). mp:128-130°C.
- Preparation of (S)-benzyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy -4-phenylbutyryl)-4-methylpentanoate (7): Boc-AHPA (6 g, 20.3 mmol) was dissolved in dichloromethane, and followed by the addition of HOBt (3 g, 22.3 mmol) and EDCI (4.5 g, 22.3 mmol) under ice bath. And after 0.5 h, L-leucine benzyl ester toluene-4-sulfonate (8.5 g, 22.3 mmol) and 3.1 mL of triethylamine were added into the solution. The mixture was reacted for 5h after the ice bath was removed. The reactant solution was washed with 10% citric acid, saturated NaHCO3 and saturated NaCl for 3 times respectively, then dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to achieve a yellowish white solid 7 (4.8 g, 47.5%).
- Preparation of (2S)-benzyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-4-phenyl -2-(2-(2H)-tetrahydropyranoxy)butyryl)-4-methylpentanoate (8): 7 (12 g, 24 mmol) was dissolved in dry dichloromethane, followed by addition of PPTS (0.6 g, 2.4 mmol) and DHP (3.6 g, 43.2 mmol) dropwise. The mixture was reacted at 35°C for 24 h until TLC test gave the reaction was accomplished, 0.5g of K2CO3 were added, and the mixture was stirred for 20 min, then the solution was washed with saturated NaCl for three times. After dried with anhydrous sodium sulfate, filtered it and evaporated the solvent under reduced pressure to get yellowish white solid 8 (12.6 g, 90.8%).
- Preparation of (2S)-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-4-phenyl-2-(2-(2H)-tetrahydropyranoxy)butyryl)-4-methylpentanoate (9): 8 (12.6 g, 21.6 mmol) was dissolved in methanol, then 1.3g Pd/C (10%) was added in several times. The air in the reactant bottle was removed out and was filled with hydrogen by a hydrogen balloon, after being reacted for 12h, filtered the mixture with two layers of filter papers. The solvent was evaporated until the residue was dried to get colorless foam like solid 9 (10.2 g, 96.0%).
- Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-4-phenyl-2-(2-(2H)-tetrahydropyran oxy)acyl)-4-methylpentanoate (10): 9 (6 g, 20.3 mmol) was dissolved in re-distilled dichloromethane, followed by addition of HOBt (3.6 g, 26.92 mmol) and EDCI (5.2 g, 26.92 mmol) under ice bath. After 0.5 h, 2 (4.3 g, 26.92 mmol) in acetonitrile solution and 3.7mL of triethylamine were added into the solution, and the mixture was reacted for 20 h after the ice bath was removed. The reactant solution was washed with 10% citric acid, saturated NaHCO3 and saturated NaCl for three times, and dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get yellow oil, and separated the oil by using flash column chromatography to obtain colorless oil 10 (8.8 g, 67%).
- Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-(amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate hydrochloride (6(BC-01)): 10 (8.8g, 13.9 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get white solid 6(BC-01) (5.94g, 88%). ESI-MS m/z:451.5 (M+H)+, 1H-NMR(600 MHz DMSO): δ 0.85-0.89 (m, 6H), 1.51-1.56 (m, 1H), 1.59-1.66 (m, 2H),2.87-2.94 (m, 2H), 4.01-4.05 (m, 2H), 4.24-5.28 (m, 1H), 5.60-5.63 (m, 2H), 6.75 (s, 1H), 7.23-7.32 (m, 5H), 8.09 (s, 3H), 8.16(d, J=6.6 Hz, 1H), 8.48(d, J=7.2 Hz, 1H), 12.05 (s, 1H). mp:128-129°C.
- Preparation of 2-((tert-butoxycarbonyl)amino)acetic acid (12): L -Glycine (2.3 g, 30 mmol) was dissolved in 1mol/L NaOH, and (Boc)2O (7.2 g, 33 mmol) in tetrahydrofuran solution was added into the mixture dropwise while stirring mechanically under ice bath. After finishing adding and for 0.5 h, the ice bath was removed. The pH of the reactant solution was retained at 10 by using 2 mol/L NaOH, and then reacted overnight. After tetrahydrofuran was evaporated, the residue was extracted with petroleum ether for 3 times, and the pH of water phase was adjusted to 2-3 by using 3 mol/L HCl, and then extracted it with ethyl acetate, dried with anhydrous sodium sulfate. After filtering, evaporating and being dried for 24 h in vacuum, a white solid 12 (5.1g, 96%) was obtained.
- Preparation of (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1-(2H))methyl-2-((tert-butoxycarbonyl)amino)acetate (13). 12 (1.1 g, 6.3 mmol) was dissolved in re-distilled dichloromethane, followed by addition of HOBt (1.2 g, 8.8 mmol) and EDCI (1.7 g, 8.8 mmol) under ice bath. After 0.5 h, 2 (1.4 g, 8.8 mmol) in acetonitrile solution and 1.2 mL of trimethylamine were added into the solution, and the mixture was reacted for 20h after the ice bath was removed. The reactant solution was washed with 10% citric acid, saturated NaHCO3 and saturated NaCl for three times, and dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get yellow oil, and separated the oil by using flash column chromatography to obtain colorless oil 13 (1.3 g, 67%).
- Preparation of (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1-(2H))methyl-2-amino)acetate hydrochloride (14): 13 (1.3 g, 4.2 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and the solution was reacted at room temperature for 3 h. Filtered it to give a white solid 14 (0.93 g, 88%).
- Preparation of (6R,7S,10S)-(5-fluoro-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-3-yl) methyl-6-benzyl-10-isobutyl-2,2-dimethyl-4,8,11-trioxo-7-(2-2H-tetrahydropyran oxy)-3-oxo-5,9,12-triazatetradecanoate (15): 9 (1.21 g, 2.45 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.65 g, 3.4 mmol) and HOBt (0.46 g, 3.4 mmol) under ice bath, and after 0.5 h, 14 (0.93 g, 3.4 mmol) and 0.48 mL of triethylamine were added into the solution. The mixture was reacted for 5h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 15 (0.86 g, 51 %).
- Preparation of (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((S) -2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)acetate hydrochloride (16(BC-02)): 15 (0.86 g, 1.24 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get a white solid 16(BC-02) (0.57 g, 85%). ESI-MS m/z:508.4 (M+H)+, 1H-NMR(400 MHz DMSO): δ 0.86-0.89 (m, 6H), 1.49-1.53 (m, 2H), 1.62-1.66 (m, 1H), 2.87-2.97 (m, 2H), 3.55-3.57 (m, 1H), 3.80-3.92 (m, 2H), 4.00-4.06 (m, 1H), 4.31-4.32 (m, 1H), 5.57-5.80 (m, 2H), 6.73 (s, 1H), 7.25-7.37 (m, 5H), 8.02-8.08 (m, 4H), 8.14(d, J=6.6 Hz, 1H), 8.59-8.62 (m, 1H), 12.01 (s, 1H). mp:136-137°C.
- The following Compounds were prepared as the general procedure as described in BC-02. (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((S)-2-((2S,3R)-3-ami no-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)propanoate hydrochloride (BC-03). ESI-MS m/z:521.5 (M+H)+, 1H-NMR(400 MHz DMSO): δ 0.85-0.91 (m, 6H), 1.25-1.28 (m, 3H), 1.43-1.51 (m, 2H), 1.60-1.64 (m, 1H), 2.87-2.94 (m, 2H), 3.53-3.57 (m, 1H), 3.99-4.04 (m, 1H), 4.24-4.31 (m, 2H), 5.54-5.64 (m, 2H), 7.27-7.34 (m, 5H), 7.98-8.00 (m, 4H), 8.12(d, J=6.6 Hz, 1H), 8.61 (d, J=6.8 Hz, 1H), 11.99 (s, 1H). mp: 111-112°C.
- (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-3-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)propanoate hydrochloride (BC-04). ESI-MS m/z:522.5 (M+H)+, 1H-NMR(400 MHz DMSO): δ 0.84-0.88 (m, 6H), 1.45-1.59 (m, 4H), 2.86-2.97 (m, 2H), 3.20-3.31 (m, 2H), 3.56-3.57 (m, 1H), 4.00-4.06 (m, 1H), 4.18-4.23 (m, 1H), 5.55-5.60 (m, 2H), 7.26-7.36 (m, 5H), 7.99-8.01 (m, 4H), 8.09-8.11(m, 1H), 8.21-8.24 (m, 1H), 11.96 (s, 1H). mp: 119-120° C.
- (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-4-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)butanoate hydrochloride (BC-05) ESI-MS m/z:536.4 (M+H)+, 1H-NMR(400 MHz DMSO): δ 0.85-0.89 (m, 6H), 1.48-1.65 (m, 6H), 2.32-2.36 (m, 2H), 2.89-2.94 (m, 2H), 3.02-3.07 (m, 2H), 3.56-3.57 (m, 1H), 4.01-4.04 (m, 1H), 4.20-4.26 (m, 1H), 5.56-5.62 (m, 2H), 7.27-7.35 (m, 5H), 7.99-8.04 (m, 5H), 8.12-8.15 (m, 1H), 11.96 (s, 1H). mp: 117-118°C.
- (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-6-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)hexanoate hydrochloride (BC-06) ESI-MS m/z:564.5 (M+H)+, 1H-NMR(400 MHz DMSO): δ 0.86-0.89 (m, 6H), 1.16-1.24 (m, 2H), 1.36-1.38 (m, 2H), 1.46-1.52 (m, 5H), 2.28-2.32 (m, 2H), 2.89-3.02 (m, 4H), 3.55 (s, 1H), 3.99-4.04 (m, 1H), 4.22-4.26 (m, 1H), 5.56 (s, 2H), 7.27-7.35 (m, 5H), 7.99-8.15 (m, 6H), 11.96 (s, 1H). mp:98-99°C.
- (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-1-((S)-2-((2S,3R)-3-ami no-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)-2-tetrahydropyrrolidine carboxylate hydrochloride (BC-07). ESI-MS m/z:548.4 (M+H)+, 1H-NMR(400 MHz DMSO): δ 0.87-0.92 (m, 6H), 1.39-1.41 (m, 1H), 1.46-1.49 (m, 1H), 1.65-1.71 (m, 1H), 1.85-1.99 (m, 2H), 2.16-2.17 (m, 1H), 2.89-2.91 (m, 2H), 3.48-3.53 (m, 4H), 3.70-3.72 (m, 1H), 3.96-4.04 (m, 1H), 4.31-4.34 (m, 1H), 4.46-4.53 (m, 1H), 5.54 (d, J=10.3 Hz, 1H), 5.67 (d, J=10.3 Hz, 1H), 6.71 (s, 1H), 7.27-7.36 (m, 5H), 8.02-8.03 (m, 3H), 8.11-8.14 (m, 2H), 12.00 (s, 1H). mp:140-141°C.
- Preparation of ( S )-5 fluorophenyl-2-((tert-butoxycarbonyl)amino)-4-methy-pentanoate (18): Boc-L-leucine (1.17g, 5 mmol) was dissolved in anhydrous tetrahydrofuran, followed by addition of pentafluorophenol (1.01g, 5.5 mmol) and EDCI (1.05g, 5.5 mmol), and the mixture was reacted at room temperature for 12 h. Evaporated the solvent and its residue was separated by column chromatography to get colorless oil 18 (1.7 g, 86%).
- Preparation of (S)-1-((2-(tert-butoxycarbonylamino)-4-methylvaleryl)oxy)urea (19): Hydroxyurea (0.35 g, 4.3 mmol) was dissolved in DMF, followed by addition of 0.52 mL of N-Methylmorpholine and 18 (1.7 g, 4.3 mmol) was added dropwise in the reactant solution, then the mixture was reacted for 12h at room temperature. Evaporated the solvent and its residue was separated by column chromatography to get white solid 19 (0.75 g, 60%).
- Preparation of (S)-1-((2-amino-4-methylvaleryl)oxy)urea hydrochloride (20): 19 (0.75 g, 2.58 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get a white solid 20 (0.47 g, 81%).
- Preparation of (2R,3S)-3-(tert-butoxycarbonylamino)-2-hydroxy-N-((S)-4-methylvaleryl)oxy)ureido)-4-phenylbutanamide (21): Boc-AHPA (1 g, 3.38 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.71 g, 3.72 mmol) and HOBt (0.5 g, 3.72 mmol), and after 0.5 h, 20 (0.84 g, 3.72 mmol) and 0.54 mL of triethylamine were added into the solution. The mixture was reacted for 5h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 21 (0.71 g, 45%).
- Preparation of (2S,3R)-3-amino-2-hydroxy-N-((S)-4-methylvaleryl)oxy)ureido) -4-phenylbutanamide hydrochloride (22): 21 (0.71 g, 1.52 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get a white solid 22 (0.48 g, 78%). ESI-MS m/z:367.3 (M+H)+, 1H-NMR(600 MHz DMSO): δ 0.80-0.92 (m, 6H), 1.56-1.75 (m, 3H), 2.90-3.05 (m, 2H), 4.01-4.06 (m, 2H), 4.53 (m, 1H), 6.48-6.55 (m, 2H), 6.88 (s, 1H), 7.26-7.38 (m, 5H), 8.02-8.09 (m, 3H), 8.63(d, J=7.2 Hz, 1H), 9.77 (m, 1H). mp:110-112°C.
- Preparation of Boc-epirubicin (24): 23 (1.09 g, 2 mmol) was dissolved in anhydrous dichloromethane, followed by addition of 0.84 mL of trimethylamine and (Boc)2O (0.52 g, 2.4 mmol) in dichloromethane solution was added into the mixture dropwise, the mixture was reacted overnight at room temperature until TLC test gave the reaction was accomplished. After the reaction was accomplished, the reactant solution was washed with 10% citric acid and saturated NaCl for 3 times, dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get compound 24 (1.09 g, 85%).
- Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino) -5-hydroxy-6-methyl-2(2H )-tetrahydropyranyloxy)-2,5,12-trihydroxy-7-methoxy-6,1 1-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl))-2-ethoxy-2-(benzyloxycarbonylamino)-4-methylpentanoate (25): Cbz-L-leucine (0.5 g, 1.87 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.36 g, 1.87 mmol) and HOBt (0.25 g, 1.87 mmol) under ice bath, and after 0.5 h, 24 (1.09 g, 1.7 mmol) was added into the solution. The mixture was reacted for 5h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 25 (0.76 g, 50%).
- Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino) -5-hydroxy-6-methy-2(2H)-tetrahydropyranyloxy)-2,5,12-trihydroxy-7-methoxy-6,11 -dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl))-2-ethoxy-2-amino-4-methylpentanoate (26): 25 (0.76 g, 0.85 mmol) was dissolved in methanol, then 0.1g Pd/C (10%) was added in several times. The air in the reactant bottle was removed out and was filled with hydrogen by a hydrogen balloon, after being reacted for 12h, filtered the mixture with two layers of filter papers. The solvent was evaporated until the residue was dried to get colorless foam like solid 26 (0.6g, 93.0%).
- Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino) -5-hydroxy-6-methyl-2(2H)-tetrahydropyranyloxy)-2,5,12-trihydroxy-7-methoxy-6,1 1-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl))-2-ethoxy-2-((2S,3R)-3-((tert-butoxycar bonyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate (27): Boc-AHPA (0.27 g, 0.88 mmol) was dissolved in anhydrous dichloromethane,, followed by addition of EDCI (0.17 g, 0.88 mmol) and HOBt (0.27 g, 0.88 mmol) under ice bath, and after 0.5 h, 26 (0.6 g, 0.8 mmol) was added into the solution. The mixture was reacted for 5h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 27 (0.47 g, 57%).
- Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino) -5-hydroxy-6-methyl-2(2H )-tetrahydropyranyloxy))-2,5,12-trihydroxy-7-methoxy-6,1 1-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl)-2-ethoxy-2-((2S,3R)-3-amino-2-hydroxy -4-phenylbutyryl)-4-methylpentanoate hydrochloride (28): 27 (0.47 g, 0.46 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get white solid 28 (0.36 g, 87%). ESI-MS m/z:833.7 (M+H)+, 1H-NMR(600 MHz DMSO): δ 0.81-0.94 (m, 6H), 1.23(d, J=7.2 Hz, 3H), 1.58-1.83 (m, 3H), 1.75-1.79 (m, 1H), 2.07-2.09 (m, 1H), 2.20-2.24 (m, 2H), 2.93-3.18 (m, 4H), 3.44-3.49 (m, 2H), 3.88(s, 3H), 3.98(t, J=5.4 Hz, 1H), 4.06-4.09 (m, 2H), 4.55 (m, 1H), 4.58-4.69 (m, 2H), 4.91-4.98 (m, 2H), 5.05-5.09(m, 1H), 5.28-5.35 (m, 1H), 5.47 (s, 1H), 5.76 (s, 1H), 6.93 (s, 1H), 7.26-7.38 (m, 5H ), 7.69(m, 1H), 7.91-7.98(m, 2H), 8.11-8.19 (m, 3H), 8.63(d, J=7.2 Hz, 1H). mp:136-138°C.
- Preparation of (S)-2-(4-(6-(5-(2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-(tert-butoxycarbonylamino)-4 -methylpentanoate (29): Boc-L-leucine (0.25 g, 1.1 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.21 g, 1.1 mmol) and HOBt (0.15 g, 1.1mmol) under ice bath, and after 0.5 h, Dasatinib (0.51 g, 1 mmol) and 0.2 mL of triethylamine were added into the solution. The mixture was reacted for 5h at room temperature after the ice bath was removed. After the reaction was accomplished, the organic layer was washed with water, 1 M citric acid, saturated sodium bicarbonate and saturated sodium chloride solution respectively, then dried with anhydrous sodium sulfate, filtered it and evaporated the solvent to get white solid 29 (0.28 g, 40%).
- Preparation of (S)-2-(4-(6-(5-(2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-amino-4-methylpentanoae hydrochloride (30): 29 (0.7 g, 1 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get white solid 30 (0.54 g, 80%).
- Preparation of (S)-2-(4-(6-(5-((2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-((2S,3R)-3-((tert-butoxycarbo nyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate (31): Boc-AHPA (0.16 g, 0.55mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.11 g,0.55 mmol) and HOBt (0.08 g, 0.55 mmol) under ice bath, and after 0.5 h, 30 (0.34 g, 0.5 mmol) was added into the solution. The mixture was reacted for 5h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 31 (0.22 g, 51%).
- Preparation of (S)-2-(4-(6-(5-(2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-((2S,3R)-3-amino-2-hydroxy-4 -phenylbutyryl)-4-methylpentanoate hydrochloride (32): 31 (0.22 g, 0.25 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2h. Filtered it to get white solid 32 (0.18 g, 84%). ESI-MS m/z:777.5 (M+H)+, 1H-NMR(600 MHz DMSO): δ 0.83-0.95 (m, 6H), 1.59-1.85 (m, 3H), 2.13(s, 3H), 2.26(s, 3H), 2.54-2.58 (m,6H), 2.90-3.05 (m, 2H), 3.60-3.70 (m, 6H), 4.01-4.06 (m, 2H), 4.53-4.58 (m, 1H), 6.09 (s, 1H), 6.88 (s, 1H), 7.26-7.38 (m, 7H), 7.48 (d, J=7.2 Hz, 1H), 8.02-8.09 (m, 3H), 8.22 (s, 1H), 8.63(d, J=7.2 Hz, 1H), 9.57-9.59 (m, 2H), 9.77-9.78 (m, 2H). mp:174-176°C.
- Aminopeptidase N and L-Leu-p-nitroanilide as substrate were purchased from Sigma cooperation.
- Preparation of buffer solution: 12.89 g Na2HPO4•12H2O and 2.18 g NaH2PO4•2H2O were dissolved in a 1000 mL volumetric flask, then the mixture was diluted to 1000 mL with fresh distilled water to obtain 50 mM Phosphate Buffer solution (PBS, pH 7.2), which was kept at room temperature to spare.
- Aminopeptidase N was dissolved in the buffer solution to obtain 0.1IU/mL solution.
- The substrate was dissolved in DMSO to obtain a 16 mmol/mL solution, which was kept in refrigerator to spare.
-
No. Aminopeptidase N solution (µL) substrate solution (µL) buffer solution (µL) Inhibitor 100 % control group 10 5 185 0 Blank control group 0 5 195 0 Inhibitor group 10 5 145 40 - The
aminopeptidase N solution 10 µL, thesubstrate solution 5 µL and compound withdifferent concentration 40 µL were added into a 96-well cell plate, and then adjusted to 200 µL with phosphate buffer saline solution (pH 7.2). 100% group did not include inhibitor. And thesubstrate solution 5 µL of blank group was adjusted to 200 µL with buffer solution. The mixture was incubated at 37 °C for 0.5h, and absorbance was then determined at 405 nm wavelength. Inhibitory ratio can be calculated as follows: - According to concentration of compound and corresponding inhibitory ratio, the IC50 value was calculated by using a fitting curve of Origin 7.5 software.
- The inhibition activity against aminopeptidase N of the compound represented by formula (I) of the present invention and ubenimex as positive control drug are shown in the following table:
Compd(compound) IC50(µM) APN 6(BC-01) 6.98 16(BC-02) 0.38 BC-03 0.36 BC-04 1.30 BC-05 1.39 BC-06 4.66 BC-07 0.15 22 2.92 28 5.56 32 7.88 ubenimex 5.12 - The result of inhibition activity against enzyme in vitro showed that, the target compounds BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, 22, 28 and 32 represented by formula (I) of the present invention all established an inhibitory activity against aminopeptidase N, wherein Compounds BC-02, BC-03, BC-04, BC-05, BC-06 and BC-07 were much more potent than the positive control drug ubenimex, especially, BC-07 showed great activity with 0.15µM. Compounds BC-01, 22, 28 and 32 showed a comparable activity with ubenimex. This indicated that synergetic fragment linked at carboxyl group of ubenimex may increase its activity against enzyme without destroying inhibition activity against enzyme of the whole structure.
- Human leukemia cell line K562, clear-cell ovarian carcinoma cell line ES-2, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, cell line Hela, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO were transferred into a culture flask and were cultured in a medium at 37°C in a 5% CO2 and saturation humidity. One bottle of cells in logarithmic growth phase was picked out and was blew and beaten to be uniform with a pipette, then took some cell suspension solution to prepare blood counting plate smear to count cells under inverted microscope, and then adjusted the cell number to be 1×105/mL by adding some culture medium. Cells were seeded on a 96-well plate on which also drug tests performed. Blank control group, negative control group, positive control group and drug test group were established on the plate while the surrounding wells of the plate were not used (but filled with sterile PBS), wherein the blank control groups were prepared by addition of culture medium 150 µL per well, the negative control groups were prepared by addition of cell suspension solution 100µL per well and
culture medium 50 µL per well, the positive control groups were prepared by addition of cell suspension solution 100µL per well and positive control drug solution 50µL per well, the drug test groups were prepared by addition of cell suspension solution 100µL per well and test compound solution 50µL per well, and there were 5 different concentrations of drug in the positive control group and the drug test group respectively: 0.01, 0.1, 1, 10, 100µmol·L-1, each concentration was established with 3 parallel wells. After the drug was added, the 96-well plate was incubated for 48 h at 37°C in a CO2 incubator of 5% CO2 and saturation humidity. Upon completion of the incubation, 20 µL MTT solution (concentration was 5mg/mL) was added to each well and incubated for an additional 4h, and after the 96-well plate was centrifuged at 2000 rpm for 30 min, medium of each well was suck out carefully and removed and 100 µL DMSO was added instead, followed by vibrating on a tablet shaker for 15min to re-dissolve formazan crystals completely. OD value of each well was measured at 570 nm wavelength using an enzyme-linked immunosorbent assay reader to calculate cellular proliferation inhibitory ratio of each drug in different concentrations. And half-inhibitory concentration (IC50) was calculated using statistical software SPSS 16.0, wherein the cellular proliferation inhibitory ratio can be calculated as following equation: - The results of proliferation inhibition activity of target compounds BC-01, BC-02, 22, 28, 32 and positive control drugs ubenimex and 5-fluorouracil (5-FU) against clear-cell ovarian carcinoma cell line ES-2, human leukemia cell line K562, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, cell line Hela, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO are shown in following table:
Compd EC50(µM) ES-2 K562 PC-3 MCF-7 Hela H7402 3-AO 6(BC-01) 2.53 5.82 10.71 1.35 0.73 1.86 2.59 BC-02 1.34 ND 3.45 ND ND 10.98 ND 22 5.66 6.52 13.34 6.74 5.55 7.98 9.06 28 3.34 4.35 7.67 2.55 1.99 4.02 3.02 32 6.45 6.33 14.13 3.65 3.23 6.75 4.89 5-FU ND 16.03 15.70 76.92 132.99 385.38 60.81 Uubenimex >400 >400 >400 32.70 79.59 188.54 >400 ND: not determined. - The results of bioactivities showed that, the target compounds BC-01, BC-02, 22, 28 and 32 represented by formula (I) of the present invention established an obvious anti-proliferation effect against all the tumor cells above. By comparing with the positive drugs ubenimex and 5-fluorouracil (5-FU), BC-01 showed much higher proliferation inhibition activity against cell line Hela, and had an obvious proliferation inhibition activity against clear-cell ovarian carcinoma cell line ES-2, human leukemia cell line K562, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO as well; BC-02 established a great proliferation inhibition activity against clear-cell ovarian carcinoma ES-2 cell line. Compounds 22, 28 and 32 were much more potent than 5-FU on proliferation inhibition activity.
- The results of cells test in vitro showed that, the target compounds BC-01, BC-02, 22, 28 and 32 represented a great proliferation inhibition activity against 7 cell lines.
- Extracted the ascites from H22 ascites tumour bearing mice, washed the ascites with sterile PBS for three times and then resuspended the cells with sterile PBS (cells counts: 3.75×107 cells/mL), inoculated it into right oxter of Kunming mice 100uL per mouse. After getting rid of the Kunming mice overweight or underweight, the tumor-bearing mice were randomized to several groups, and then the mice was began to administrate drug based on administration strategy, henceforth administrated once a day with two days off when having administrated for 5 days until two cycles one of which had 7 days (the volume of administration was: 200uL/20g once per mouse, the method of administration was: oral gavage). The body weight of mouse was recorded at the beginning and ending of every cycle, and the average volume of administration was 200uL/20g once per mouse; the method of administration was: oral gavage, and administrated once a day.
- The H22 tumor bearing mice were randomized to 4 groups (10 mice per group) after being weighted. (1) the negative control: PBS; (2) Xeloda group: 100mg/kg/d; (3) TFU group: 70mg/kg/d; (4) BC-01 group: 100mg/kg/d. And administrated once a day with two days off when having administrated for 5 days until two cycles one of which had 7 days (the volume of administration was: 200uL/20g once per mouse, the method of administration was: oral gavage). The size of tumor was measured by using a vernier calliper and weight of mouse by using an electronic balance at the beginning and ending of every cycle, then calculated their mean value. The mice were sacrificed after 13 days and the tumors were taken out and weighted. Tumor volume and inhibitory ratio were calculated as the following equation (L and W referred to length and width of tumor respectively):
- The H22 tumor-bearing mice were randomized to 4 groups (7 mice per group) after being weighted. (1) the negative control: PBS; (2) 5-FU group: 20mg/kg/d; (3) BC-01 group: 50mg/kg/d; (4) BC-01 group: 70mg/kg/d; (5) BC-02 group: 70mg/kg/d; (6) combination group of ubenimex and 5-FU: 5-FU: 15mg/kg/d (intravenous injection), ubenimex: 30mg/kg/d (oral administration). And administrated once a day with two days off when having administrated for 5 days until two cycles one of which had 7 days (the volume of administration was: 200uL/20g once per mouse, the method of administration was: tail intravenous injection). The size of tumor was measured by using a vernier calliper and weight of mouse by using an electronic balance at the beginning and ending of every cycle, then calculated their mean value. The mice were sacrificed after 13 days and the tumors were taken out and weighted. Tumor volume and inhibitory ratio were calculated as the following equation (L and W referred to length and width of tumor respectively):
-
Ctrl (control group) Xeloda TFU BC-01 tumor weight 0.79 0.60 0.68 0.26 SD 0.52 0.25 0.45 0.13 inhibitory ratio (%) 24.53 13.38 66.67 -
Compd Number of mice alive Body weight(g) Tumor weight(g) inhibitory ratio (%) beginning(g) ending(g) Ctrl(control group) 7 31.60±4.33 33.61±4.21 1.37±0.27 5-FU 7 31.37±4.05 31.92±4.98 0.84±0.19 38.83 BC-01(50) 7 30.32±1.57 31.53±4.71 0.58±0.12 57.66 BC-01(70) 7 30.00±1.15 31.05±1.78 0.37±0.08 73.00 BC-02 7 31.81±4.99 32.83±4.64 0.63±0.01 54.31 5-FU+Bes 7 32.96±1.75 34.54±1.18 0.47±0.19 65.55 -
Group Administration dosage Method of administration Number of mice Ctrl group Equivalent PBS solution Intravenous injection 8 Xeloda 108mg/kg/d (0.3mmol/kg/d) Oral gavage 8 BC-01 120mg/kg/d (0.25 mmol/kg/d) Oral gavage 8 BC-02 130 mg/kg/d (0.25 mmol/kg/d) Oral gavage 8 5- FU 20 mg/kg/d (0.15 mmol/kg/d) Intravenous injection 8 BC-01 50 mg/kg/d (0.1 mmol/kg/d) Intravenous injection 8 BC-07 58.3 mg/kg/d (0.1 mmol/kg/d) Intravenous injection 8 BC-02 55mg/kg/d (0.1 mmol/kg/d) Intravenous injection 8 BC-02 79 mg/kg/d (0.15 mmol/kg/d) Intravenous injection 8 - The ascites was extracted from H22 tumour bearing mice with well growth, followed by adding sterile PBS to dilute the ascites to a concentration of 8.5×107 cells/mL. 100uL of the cells solution was suck up by a 1mL sterile injector to inoculate into right oxter of mice. After 3 days, the mice were weighted and randomized to several groups (almost 8 mice per group, and a bit more in control group). These mice were administrated a predetermined dosage by intravenous or oral gavage with two days off when having administrated for 5 days until two cycles one of which had 7 days. The body weight of mouse was recorded at the beginning and ending of every cycle,
- After being administrated for two cycles, the mice were weighted and recorded, then sacrificed by cervical dislocation to get lung, liver and spleen weighted respectively.
- Overall difference was calculated by the function of one-way analysis of variance (One-Way ANOVA) in Origin 7.5 software; and t-test was used for pair-comparison between drug administrated group and blank group, and the inhibitory ratio of drug in each group was calculated based on the following equation:
-
Groups Number of mice alive Body weight(g) Tumor weight(g) Tumor growth inhibition (%) Beginning(g) Ending(g) Ctrl group 8 29.66±3.60 31.37±4.97 0.60±0.20 *Xeloda 7 33.00±1.81 31.42±3.56 0.35±0.09 40.95 *BC-01(50) 7 27.87±2.55 29.50±2.15 0.12±0.16 79.52 *BC-02(70) 7 27.98±3.73 27.49±4.94 0.06±0.11 89.76 5-FU 4 26.89±2.06 25.51±4.50 0.54±0.06 10.41 BC-01(50) 8 31.52±3.63 31.78±5.76 0.09±0.14 84.38 BC-07 6 32.17±3.86 31.44±5.01 0.34±0.24 43.61 BC-02 7 25.67±2.62 28.35±3.71 0.06±0.11 89.52 BC-02 7 30.32±4.42 31.13±3.55 0.0±0.0 100 - As observed in
figures 11-14 , compounds BC-01 and BC-02 all showed excellent anti-tumor growth activity, what's more, at a dosage of 0.15mmol/kg/d of intravenous administrated BC-02, no tumor appeared until the end of the experiment. - At the end of experiments, there is no significant changes in visceral organs by visual observation of dissected mice, it indicates that compounds at administration dosage have no evident toxicity.
Claims (20)
- A multi-targeted Ubenimex pro-drug derivative represented by general structural formula (I), as well as an enantiomer, a diastereoisomer, a racemate or a pharmaceutically acceptable salt or solvate thereof:
R3 represents: - The multi-targeted Ubenimex pro-drug derivative according to claim 1, wherein in the general structural formula (I), R represents the following substituents:
- A preparation method of the multi-targeted Ubenimex prodrug derivative according to claim 2, includes: 5-fluorouracil is reacted with an aldehyde 1-1 to provide intermediate 2', intermediate 2' is reacted with Boc-L-leucine via condensation reaction to give intermediate 3', protecting group of intermediate 3' is removed to provide compound 4', compound 4' is reacted with Boc-AHPA via condensation reaction to give compound 5', compound 5' is deprotected to give target compound 6'; synthesis route thereof is as follows:
- An alternative preparation method of the multi-targeted Ubenimex pro-drug derivatives according to claim 2, includes: 5-fluorouracil is reacted with an aldehyde 1-1 to provide intermediate 2'; hydroxyl group of compound 7 is protected by DHP to give intermediate 8, benzyl group of intermediate 8 is removed to provide compound 9, compound 9 is condensed with intermediate 2' to give compound 10', compound 10' is deprotected via acid to provide target compound 6'; synthesis route thereof is as follows:
- A preparation method of the multi-targeted Ubenimex pro-drug derivative according to claim 3, includes: compound 14' or its cationic salt is condensed with compound 9 via EDCl and HOBt in anhydrous DCM to give compound 15', compound 15' is deprotected via acid HCl-AcOEt to give target compound 16'; synthesis route thereof is as follows:
- The method according to claim 10, wherein when Y represents NH and M represents
- A preparation method of the multi-targeted Ubenimex pro-drug derivative according to claim 4, includes: Boc-L-Leucine 17 is condensed with pentafluorophenol to give intermediate 18, intermediate 18 is reacted with hydroxyurea to provide compound 19, protecting group of compound 19 is removed via acid to give compound 20, compound 20 is condensed with Boc-AHPA to give compound 21, compound 21 is deprotected to provide target compound 22; synthesis route thereof is as follows:
- A preparation method of the multi-targeted Ubenimex pro-drug derivative according to claim 5, includes: Epirubicin is protected via (Boc)2O to give compound 24, compound 24 is condensed with Cbz-L-leucine to give compound 25, amino-protecting group of compound 25 is removed to give compound 26, compound 26 is condensed with Boc-AHPA to give compound 27, compound 27 is deprotected to give target compound 28; synthesis route thereof is as follows:
- A preparation method of the multi-targeted Ubenimex pro-drug derivatives according to claim 6, includes: Boc-L-leucine 17 is condensed with compound A-E-H to give compound 29', compound 29' is deprotected via acid to give compound 30', compound 30' is condensed with Boc-AHPA to give compound 31', compound 31' is deprotected to give target compound 32'; synthesis route thereof is as follows:
- A preparation method of the multi-targeted Ubenimex pro-drug derivative according to claim 7, includes: compound 9 is condensed with compound Q-Y-H to give target compound 33'; synthesis route thereof is as follows:
- A preparation method of the multi-targeted Ubenimex pro-drug derivative according to claim 7, includes: compound 9 is condensed with p-hydroxybenzaldehyde to give compound 33, compound 33 is reduced to give alcohol 34, alcohol 34 is reacted with triphosgene to give compound 35, compound 35 is reacted with H-R4 to form compound 36, compound 36 is deprotected to give target compound 37; synthesis route thereof is as follows:
- Use of the multi-targeted Ubenimex pro-drug derivative according to any one of claims 1 to 7 for manufacturing a drug of treating or preventing a variety of tumors.
- The use of the multi-targeted Ubenimex pro-drug derivative for manufacturing a drug of treating or preventing a variety of tumors according to claim 17, wherein the tumors represent solid tumors resistant to chemotherapy drugs.
- Use of the multi-targeted Ubenimex pro-drug derivative according to any one of claims 1 to 7 for treating or preventing a variety of tumors.
- The use of the multi-targeted Ubenimex pro-drug derivative for treating or preventing a variety of tumors according to claim 19, wherein the tumors represent solid tumors resistant to chemotherapy drugs.
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CN104888189B (en) * | 2015-06-12 | 2017-09-29 | 上海来益生物药物研究开发中心有限责任公司 | The new opplication of the phenyl valyl isoleucine analogues of 3 amino, 2 hydroxyl 4 |
EA201891235A1 (en) * | 2015-11-25 | 2018-12-28 | Дзе Риджентс Оф Дзе Юниверсити Оф Калифорния | COMPOSITIONS OF MEDICINES FOR THE TREATMENT OF MALIGNANT TUMOR |
CN107174576A (en) * | 2017-06-06 | 2017-09-19 | 青岛大学附属医院 | Ubenimex is in reversing tumor cellular drug resistance application |
CN109956907A (en) * | 2017-12-23 | 2019-07-02 | 江西润泽药业有限公司 | A kind of ubenimex prodrug derivant and its preparation and use |
WO2019151320A1 (en) * | 2018-01-30 | 2019-08-08 | 国立大学法人大阪大学 | Anticancer agent |
CN109705044B (en) * | 2018-12-18 | 2021-03-23 | 江西润泽药业有限公司 | Synergistic prodrug derivative of ubenimex derivative and compound with anti-tumor activity, preparation method and application thereof |
CN109846873B (en) * | 2018-12-18 | 2022-03-18 | 江西润泽药业有限公司 | Anti-tumor medicine composition |
CN109646429B (en) * | 2018-12-21 | 2021-08-10 | 江西润泽药业有限公司 | Solid dosage form containing ubenimex prodrug derivative and preparation method thereof |
CN109528664B (en) * | 2018-12-21 | 2021-07-06 | 江西润泽药业有限公司 | Lyophilized powder containing ubenimex-antitumor drug synergistic prodrug derivative and preparation method thereof |
CN109846818B (en) * | 2018-12-21 | 2021-07-06 | 江西润泽药业有限公司 | Injection of antineoplastic medicine with ubenimex structure and preparation method thereof |
CN114349816B (en) * | 2021-11-30 | 2024-08-30 | 潍坊博创国际生物医药研究院 | Aminopeptidase N/CD 13-based small molecule coupling molecule and preparation method and application thereof |
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PT740650E (en) * | 1994-01-28 | 2004-10-29 | Univ Kentucky Res Found | CO-DRUGS AS A METHOD OF CONTROLLED DRUG ADMINISTRATION |
JP2000327568A (en) | 1999-03-17 | 2000-11-28 | Nippon Kayaku Co Ltd | Apoptosis inducer |
JP2001131066A (en) * | 1999-08-25 | 2001-05-15 | Nippon Kayaku Co Ltd | Apoptosis enhancer |
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CN101531613B (en) | 2009-04-16 | 2012-01-18 | 山东大学 | Aminopeptidase N inhibitor bestatin dino ester, synthesis and application thereof |
WO2013026942A1 (en) | 2011-08-25 | 2013-02-28 | The Provost, Fellows, Foundation Scholars, And The Other Members Of Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin | Tubulin binding agents |
JP5934986B2 (en) * | 2011-09-07 | 2016-06-15 | 公益財団法人ヒューマンサイエンス振興財団 | Apoptosis-inhibiting protein ligand-estrogen receptor ligand hybrid compound, estrogen receptor degradation inducer using the same, and preventive and therapeutic agent for breast cancer, cervical cancer or ovarian cancer |
CN103044437B (en) * | 2012-12-21 | 2015-08-26 | 上海交通大学 | Be used for the treatment of the amphipathic conjugate nano particle of tumour and preparation method, application |
CN103588713B (en) | 2013-06-07 | 2014-09-17 | 潍坊博创国际生物医药研究院 | Multi-target ubenimex prodrug derivative, as well as preparation method and application thereof |
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US9861703B2 (en) | 2018-01-09 |
CN103588713B (en) | 2014-09-17 |
EP2947070B1 (en) | 2018-09-26 |
US20160193347A1 (en) | 2016-07-07 |
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